WO2016009594A1 - Hepatoprotective agent, glucose metabolism-improving agent, and anti-obesity agent - Google Patents

Hepatoprotective agent, glucose metabolism-improving agent, and anti-obesity agent Download PDF

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WO2016009594A1
WO2016009594A1 PCT/JP2015/003141 JP2015003141W WO2016009594A1 WO 2016009594 A1 WO2016009594 A1 WO 2016009594A1 JP 2015003141 W JP2015003141 W JP 2015003141W WO 2016009594 A1 WO2016009594 A1 WO 2016009594A1
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agent
dihydroxy
methoxybenzyl alcohol
obesity
group
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PCT/JP2015/003141
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French (fr)
Japanese (ja)
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渡辺　貢
仁志 千葉
博敏 布田
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株式会社渡辺オイスター研究所
国立大学法人北海道大学
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Priority to JP2016534096A priority Critical patent/JP6978199B2/en
Publication of WO2016009594A1 publication Critical patent/WO2016009594A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/075—Ethers or acetals
    • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/618—Molluscs, e.g. fresh-water molluscs, oysters, clams, squids, octopus, cuttlefish, snails or slugs

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  • the present invention relates to a liver protective agent, a glucose metabolism improving agent and an anti-obesity agent containing 3,5-dihydroxy-4-methoxybenzyl alcohol, which is an antioxidant.
  • the level of oxidation in the living body is almost constant by the balance between the active oxygen production system and the elimination system by antioxidants, but this balance is lost due to various factors such as drugs, radiation, and ischemia, and active oxygen production Leaning toward the system is said to be oxidative stress.
  • This accumulation of oxidative stress is thought to contribute to various diseases and aging such as cancer, arteriosclerotic disease, ischemia / reperfusion injury, rheumatoid arthritis, diabetes, Alzheimer's disease and Parkinson's disease neuropathy It is.
  • antioxidants are roughly classified into two groups based on their structure.
  • examples of enzymatic antioxidants include superoxide dismutase (superoxidedismutase, SOD), catalase (catalase, CAT), glutathione peroxidase (glutathioneperoxidase, GPx), glutathione S-transferase (glutathione S-transferase, GST), glutathione reductase (glutathionereductase), Examples include peroxiredoxin (Prx).
  • non-enzymatic antioxidants include ascorbic acid, ⁇ -tocopherol, ⁇ -tocopherol, glutathione, GSH, carotenoids, flavonoids, metallothionein, etc. .
  • oysters for example, oysters (Crassostreagigas) are bivalves belonging to the order of the squirrel crabs, and their habitat extends throughout Japan and other parts of East Asia. In recent years, oysters have been cultivated in France and Australia and are famous as the most edible oysters in the world.
  • Oysters have been edible since ancient times due to their high nutritional value, but as mentioned above, they are said to contain a large amount of minerals such as calcium, zinc, selenium, copper, and manganese in addition to glycogen and protein.
  • SOD SOD
  • CAT metallothionein
  • UCP5 uncouplingprotein5
  • ascorbic acid ascorbic acid
  • the inventors of the present invention succeeded in finding an excellent so-called new antioxidant substance from oysters, further determining its chemical structure, and succeeding in chemical synthesis of the antioxidant substance.
  • the present inventors have succeeded in providing so-called novel antioxidants and antioxidant compositions which are excellent both when they are not derived from oysters or when they are derived from oysters.
  • liver protective agent having a liver protective action including an anti-apoptotic action, an anti-fatty liver action, an anti-NASH action, an anti-inflammatory action, and an anti-fibrotic action, a glucose metabolism improving agent, and an anti-obesity agent.
  • the present invention comprises 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient, It is characterized by Or 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) contained in the extract extracted from oyster meat as an active ingredient, It is characterized by Or A fraction of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was recovered from oyster meat using ethyl acetate, and the recovered 3,5-dihydroxy-4-methoxybenzyl alcohol was recovered.
  • Alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient It is characterized by Or 3,5-dihydroxy-4-methoxybenzyl alcohol fraction was recovered from oyster meat using ethyl acetate and ethanol, and the recovered 3,5-dihydroxy-4- Methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient, It is characterized by Or The oyster meat is stored in the extraction container in which the extraction solution is stored, the solution containing the oyster meat extract is collected, ethyl acetate is added to the collected solution containing the oyster meat extract, and 3,5-dihydroxy-4-methoxybenzyl The alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) fraction is recovered, and the recovered 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is used as an active ingredient.
  • the oyster meat is stored in the extraction container in which the extraction solution is stored, and the solution containing the oyster meat extract is collected. Ethyl acetate and ethanol are added to the collected solution containing the oyster meat extract, and 3,5-dihydroxy-4- The fraction of methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is recovered, and the recovered 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is an active ingredient.
  • the oyster meat is stored in the extraction container in which the extraction solution is stored, the solution containing the oyster meat extract is collected, ethanol is added to the collected solution containing the oyster meat extract, and the supernatant liquid and the precipitate are separated.
  • the separated supernatant is taken out, and ethyl acetate is added to the supernatant to separate the ethyl acetate layer and the aqueous layer,
  • the separated ethyl acetate layer solution was concentrated to recover 3,5-dihydroxy-4-methoxybenzyl alcohol fraction, and the recovered 3,5-dihydroxy-4 -Methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient, It is characterized by Or
  • the hepatoprotective agent has a hepatocyte protective effect, It is characterized by Or The hepatoprotective agent has an anti-apoptotic effect, It is characterized by Or The hepatoprotective agent has an anti-fatty liver effect, It is characterized by Or The hepatoprotective agent has an anti-NASH effect, It is characterized by Or The hepatoprotectant has an anti-inflammatory effect in the liver, It is characterized by Or The hepatoprotective agent has an
  • hepatocellular protective action, anti-antibacterial agent comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) not derived from oysters or derived from oysters as an active ingredient.
  • excellent ability to provide apoptotic action, anti-fatty liver action, anti-NASH action, anti-inflammatory action, anti-fibrosis action, glucose metabolism improving action, liver protective agent having anti-obesity action, sugar metabolism improving agent and anti-obesity agent There is an effect.
  • FIG. 6 is an explanatory diagram showing the results of analyzing changes in body weight in mice of NC group, HF ⁇ group, and HF + group from 1 to 23 weeks after the start of the experiment. It is explanatory drawing which shows the result of having analyzed the body weight and liver weight of each mouse
  • NC, HF +, HF using anti-CD3 antibody (antibody specific for lymphocytes) and anti-F4 / 80 (antibody specific for macrophages) to infiltrate lymphocytes and macrophages, which are characteristic of liver inflammatory response -It is explanatory drawing explaining the typical dyeing
  • reference numeral 1 denotes an extraction container, in which an extraction solution 2 for extracting an extract from oyster meat is stored. And the raw oyster meat 3 is accommodated in the extraction container 1 in which this extraction solution 2 is stored, and the process of extracting the extract containing various active ingredients of oyster meat is performed.
  • the extraction solution 2 containing the oyster meat 3 in the extraction container 1 was agitated to make the extraction more efficient.
  • the oyster meat 3 itself may be damaged, and it is preferable not to perform the stirring operation at the time of the extraction process.
  • the extraction solution 2 from which the oyster meat extract has been extracted as described above is then concentrated in the concentration step.
  • the ethanol solution 4 is added to the concentrated solution 6 to obtain a solution having an ethanol concentration of about 70%. Thereafter, the mixture is stirred and separated into a precipitate 7 and a supernatant 8.
  • the precipitate 7 is dried, tableted, and finally used for health food.
  • the supernatant liquid 8 may be discarded because it does not contain any active ingredient of oyster meat extract or is contained in a very small amount.
  • active ingredients relating to the oyster meat extract were also present in this supernatant liquid 8, and now this supernatant liquid 8 is used without being discarded. Yes.
  • the supernatant 8 is concentrated again and the concentrated solution is finally dried.
  • the dried product does not become a complete solid, but can be formed into a paste, and is manufactured as a paste-like health food. And this paste-form health food is melt
  • oyster meat extract containing 3,5-dihydroxy-4-methoxybenzyl alcohol which will be described later, is recovered using the supernatant 8 described above. Is.
  • the ethanol content of the supernatant 8 is removed from the supernatant 8 with an evaporator or the like, and concentrated to about a half amount.
  • a 40 mL portion of the supernatant 8 is concentrated to ensure a 20 mL supernatant 8 concentrate 9.
  • the diluted solution 10 is produced by diluting the 20 mL concentrated solution to about 5 times.
  • the amount of the diluted solution 10 is 100 mL. This process is performed in order to remove impurities as much as possible.
  • ethyl acetate 5 for example, about 200 mL of ethyl acetate 5 is added to the 100 mL of the diluted solution 10. Then, the aqueous layer 10a and the ethyl acetate layer 11 are separated by stirring or using a separator. Then, with the passage of time, this mixed solution is formed separately into the aqueous layer 10a and the ethyl acetate layer 11.
  • the 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl ⁇ alcohol) can be separated and purified from the oyster extract in any process, It was confirmed that it was present, what structure is 3,5-dihydroxy-4-methoxybenzyl alcohol, and moreover, 3,5-dihydroxy
  • 3,5-dihydroxy-4-methoxybenzyl alcohol 3,5-dihydroxy-4-methoxybenzyl alcohol
  • the oyster meat 3 is put into the extraction solution 2 containing the ethanol solution 4 to extract the oyster meat active ingredient extract (step 100, step 102).
  • the extract is concentrated (step 104). Then, for example, the ethanol solution 4 is added to the concentrated solution 6 to obtain a solution having an ethanol concentration of about 70% (step 106). Then, it stirs and isolate
  • the ethanol content is eliminated (step 110), and chloroform, ethyl acetate, and butanol are added to hexane from each hexane into the supernatant 8 diluted approximately 5 times to produce each fraction. To do.
  • concentrate to 100 mL with a rotary evaporator or the like, add 80 mL of distilled water to 20 mL of the concentrated solution, transfer to a separatory funnel, and perform hexane extraction.
  • the aqueous layer is extracted stepwise in the order of 200 mL chloroform, 200 mL ethyl acetate, and 200 mL butanol.
  • the fractions charged with the respective organic solvents are concentrated by, for example, an evaporator, and then observed by thin-layer-chromatography (hereinafter referred to as TLC; TLC: thin-layer chromatography). ) To search for antioxidant power.
  • the ethyl acetate fraction was concentrated using an evaporator and then extracted with a so-called silica open column (FIG. 28, step 1126), and the ethyl acetate / chloroform extraction fraction at a ratio of 3: 2 was selected ( 29), finally, the fraction was analyzed by HPLC (reverse phase column) to 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4- It was possible to separate and purify methoxybenzyl alcohol) (step 116).
  • HPLC reverse phase column
  • 3,5-dihydroxy-4-methoxybenzyl alcohol can also be separated and purified by the following operation.
  • Trolox (0.075 mol / L phosphate buffer 2.3, 5 mL, 6.3 x 10 -7 mol / L Fluorescein (fluorescent probe) 0.3 mL, 7% (w / v) methylated ⁇ -cyclodextrin (Wako) mixed solution) Wako) or 0.05 mL of the test sample is heated at 37 ° C. for 10 minutes.
  • the antioxidant activity is indicated by the length of time (horizontal axis) during which the fluorescence measurement value at the start of measurement (for example, the vertical axis in FIG. 27) is maintained, and the longer the time, the stronger the antioxidant activity. To do.
  • HPLC high performance liquid chromatography
  • 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is a phenolic compound as shown in FIG.
  • the antioxidant power is measured by the ORAC method.
  • ORAC AntioxidantUnit Study Group
  • the duration of the antioxidant action and its titer can be evaluated together with a single measurement, and the experimental operation is easy, so it is considered advantageous for the measurement in this example.
  • Diphenyl-1-pyrenlphosphine itself does not fluoresce, but fluoresces when oxidized.
  • DPPP diphenyl-1-pyrenlphosphine
  • the cells to which 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) in the present invention is added are cultured for 5 days, and then the cells labeled with DPPP are 2, 2 ′.
  • the fluorescence intensity of DPPP in each cell was measured.
  • the added cells had a lower fluorescence intensity depending on the concentration.
  • inhibition of oxidation by the antioxidant substance 3,5-dihydroxy-4-methoxybenzyl alcohol was observed (see FIG. 35).
  • the 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) can be efficiently extracted from the oyster meat extract according to the present invention, and thus the oyster meat extract As a result, an antioxidant composition and an antioxidant can be efficiently recovered and produced in large quantities.
  • the structure is confirmed by measuring the nuclear magnetic resonance spectrum (NMR: AMX-500, Bruker, Düsseldorf) and mass spectrum (LXQ, Thermo Scientific, Waltham) for the synthesized substance.
  • potassium carbonate (4.50 g, 32.6 mmol) was added to a dimethylformamide (DMF) solution (45 mL) of methyl gallate (5.00 g, 27.2 mmol), and the mixture was stirred at 85 ° C. for 1 hour.
  • methyl iodide (4.00g, 28.2mmol) 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient in an ice bath, was gradually added dropwise and stirred for 30 minutes, and further stirred at room temperature for 24 hours.
  • the reaction solution was filtered, purified water was added, extraction was performed with ethyl acetate, and the separated ethyl acetate layer was washed with saturated brine and dried over sodium sulfate.
  • 3,5-dihydroxy-4- methoxybenzyl alcohol an antioxidant obtained from oysters, is a highly hydroxylated aromatic compound. sell.
  • antioxidant substance identified in the present invention exhibits high antioxidant activity in cell experiments using ORAC or AAPH for observing radical scavenging ability.
  • the substance purified according to the present invention is amphiphilic, but the ORAC value was three times higher than that of L (+)-ascorbic acid, which is widely used as one of the few water-soluble antioxidants.
  • the effect of the substance as an antioxidant is highly expected.
  • the fact that the substance suppressed LDL oxidation in a dose-dependent manner suggests that the substance exerts an anti-arteriosclerotic effect through the prevention of LDL oxidation.
  • probes such as cis-parinaricacid (PnA), fluoresceinatedphosphoethanolamine, and undecylamine-fluorescein have been developed for the purpose of observing live cell oxidation in real time.
  • PnA cis-parinaricacid
  • fluoresceinatedphosphoethanolamine fluoresceinatedphosphoethanolamine
  • undecylamine-fluorescein have been developed for the purpose of observing live cell oxidation in real time.
  • PnA is often used as a probe for observing the oxidation of living cells, but PnA is often cytotoxic and has been reported to affect the physiological activity of cells.
  • DPPP used in the present invention does not affect cell proliferation or cytotoxicity for at least 3 days, and DPPP and oxidized DPPP are localized in the cell membrane of living cells and stable for at least 2 days. Has been reported. The DPPP itself does not fluoresce, but the oxidized DPPP fluoresces. Conventionally, a method for observing lipid peroxides in living cells using this DPPP has been established, and the antioxidant activity of vitamin E has been confirmed using human monocyte-based floating cells (U937).
  • NASH non-alcoholic steatohepatitis
  • a novel antioxidant was found from oysters, and its chemical structure was determined to be 3,5-dihydroxy-4-methoxybenzyl alcohol. Furthermore, the chemical synthesis method was also confirmed.
  • the ORAC value of the substance according to the present invention is 1.24 ⁇ 0.3, 5 ⁇ mol TE / ⁇ mol for the purified product, 1.47 ⁇ 0.40 ⁇ mol TE / ⁇ mol for the synthesized product, and chlorogenic acid and L (+) of the water-soluble antioxidant substance.
  • -It was an intermediate antioxidative strength of ascorbic acid.
  • the substance 3, 5-dihydroxy-4-methoxybenzyl alcohol (3, 5-dihydroxy-4- methoxybenzyl alcohol) according to the present invention shows an antioxidant ability in a dose-dependent manner, It is added that the substance showed an antioxidant ability in a dose-dependent manner in the antioxidant ability experiment using C3A cells.
  • the present inventors administered a oyster extract containing a high concentration of 3,5-dihydroxy-4-methoxybenzylmethoxyalcohol to a NASH model mouse, and the liver An experiment was conducted to determine whether or not it has a protective action.
  • FIG. 1 to FIG. 22 show experimental data for explaining the present invention.
  • the NC group of mice is fed a normal diet (MF), and the HF + group of mice is fed with a high fat diet (HFD-60) and 3,5-dihydroxy-4-methoxybenzyl.
  • MF normal diet
  • HFD-60 high fat diet
  • a diet containing 5% oyster extract containing a high concentration of alcohol was given, and the HF-group of mice was fed only a high fat diet (HFD-60). It should be noted that the food is given only for the amount that the mouse eats freely.
  • mice 4 weeks old mice are set at the start of the experiment, and 0.2 mg of oxidized LDL is injected into the tail vein every 2 days from the 20th week of the experiment. Sacrifice 12 hours after the final injection of oxidized LDL. Food is stopped 16 hours before sacrifice, anesthetized with diethyl ether, and after blood removal, a sample is taken.
  • Fig. 2 shows the analysis items.
  • liver shows neutral fat (mg / dl), total cholesterol (mg / dl), free fatty acid (mEq / L) and TBARS ( ⁇ M)
  • plasma shows AST (U / L), ALT (U / L), glucose (mg / dL), insulin ( ⁇ g / L), insulin resistance index (HOMA-IR), neutral fat (mg / dL), total cholesterol (mg / dL) And free fatty acid (mEq / L).
  • Fig. 3 shows the food intake (g) of the HF + group and HF- group of each mouse.
  • the amount of food intake is the amount obtained by measuring the amount of food initially given and the amount of food after 3 and 4 days, and subtracting the amount of food given 3 and 4 days from the amount of food initially given.
  • FIG. 3 represents the food intake for 1 to 23 weeks from the start of the experiment
  • (b) in FIG. 3 represents the lifetime food intake of each mouse in the HF + group and HF- group.
  • (a) in FIG. 3 shows that the standard time deviation is large in each mouse of the HF ⁇ group and the HF + group from the first to the tenth week from the start of the experiment. This is because the mouse puts the food outside the container. It is for putting out and spreading.
  • the lifetime food intake of each mouse in the HF + group and the HF ⁇ group is not statistically significant, and the lifetime food intake is considered to be the same.
  • Fig. 4 shows the results of analyzing the appearance of the mouse and the color of the liver.
  • FIG. 5 shows the results of analysis of changes in body weight of mice in the NC group, HF ⁇ group and HF + group for 1 to 23 weeks from the start of the experiment.
  • FIG. 6 (a) is the body weight of the mouse
  • FIG. 6 (b) is the liver weight
  • FIG. 6 (c) is the liver weight per body weight. Results are shown.
  • liver weight per body weight of the HF + group was smaller than the liver weight per body weight of the HF ⁇ group. That is, an anti-obesity action was confirmed in the HF + group of mice.
  • FIG. 7 shows score criteria in pathological observation
  • FIG. 8 shows photographs of normal cells and pathological cells in each determination.
  • score 0 corresponds to the normal cell shown in FIG. 8
  • score 2-3 corresponds to the pathological cell shown in FIG.
  • the criteria for determining the score are “Fat accumulation degree”, “Burning degree”, “Inflammation degree”, and “Fibrosis degree”.
  • the occupancy ratio that is dyed red by fat staining is obtained by image analysis software, and is determined by each occupancy ratio.
  • “Burning degree” is determined by counting how often cells swelled by HE staining are scattered in one visual field (whether they are scattered or panlobular).
  • “Inflammation” is determined by counting how many lesions are present in one field of view because infiltration of blue lymphocytes and macrophages is observed in the liver when inflammation is detected by HE staining.
  • the “fibrosis degree” is determined by Masson trichrome staining that stains the fibers blue, based on the pathological condition of whether the cell periphery and veins are connected, or whether they are connected.
  • FIG. 9 shows the results of pathological observation of the livers of mice in the NC group, the HF ⁇ group, and the HF + group.
  • FIG. 10 shows the result of analyzing the concentration of each lipid in the liver of each mouse of NC group, HF-group and HF + group.
  • FIG. 11 shows the results of analysis of AST activity and ALT activity in mouse plasma of NC group, HF ⁇ group and HF + group.
  • FIG. 12 shows the results of analysis of glucose, insulin concentration, and insulin resistance index in the plasma of each mouse in the NC group, HF ⁇ group, and HF + group.
  • Insulin resistance index fasting insulin level (mU / ml) x fasting blood glucose level (mmol / L) /22.5 The following formula is used.
  • FIG. 13 shows the result of analyzing the concentration of each lipid in the plasma of each mouse in the NC group, HF-group and HF + group.
  • FIG. 14 and FIG. 15 show genes related to the inflammatory reaction in the liver.
  • hepatocytes are inflamed by reactive oxygen species (ROS) in the liver of a NASH model mouse
  • ROS reactive oxygen species
  • macrophages and lymphocytes gather there, thereby promoting macrophage migration.
  • the cytokine IL-6 and TNF- ⁇ genes are elevated.
  • F4 / 80 and CD3 genes which are markers that specifically recognize macrophages and lymphocytes, are elevated.
  • FIG. 14 shows liver genes related to the inflammatory reaction of each mouse in the NC group, HF ⁇ group, and HF + group
  • FIG. 14A shows the TNF- ⁇ gene
  • FIG. 14B shows the IL-6 gene.
  • FIG. 14 (c) shows the result of analysis of the F4 / 80 gene, which is a macrophage marker
  • FIG. 14 (d) shows the result of analysis of the CD3 gene, which is a lymphocyte marker.
  • 16 and 17 show genes related to apoptosis in the liver.
  • FIG. 16 shows liver genes related to apoptosis in mice of NC group, HF ⁇ group and HF + group
  • FIG. 16A shows Bax gene
  • FIG. 16B shows Bcl-xl gene
  • FIG. Fig. 16 (d) shows the results of analysis for the Bcl2 gene and Fig. 16 (d) for the p53 gene.
  • the HF + group showed a decrease in Bax and an increase in Bcl2, Bcl-xl, and p53 compared to the HF-group. That is, in the liver of the HF + group, a decrease in genes related to apoptosis and an increase in genes related to anti-apoptosis were confirmed.
  • fibrosis is observed when hepatocytes are inflamed by reactive oxygen species (ROS) in the liver of a NASH model mouse.
  • ROS reactive oxygen species
  • FIG. 18 shows liver genes related to fibrosis of NC group, HF ⁇ group, and HF + group mice
  • FIG. 18A shows COL1a2 genes
  • FIG. 18B shows COL3 genes
  • FIG. ) For the COL4 gene
  • FIG. 18 (g) for the ⁇ SMA gene It is the result of analysis.
  • the HF + group showed lower COL1a2, COL3, COL4, TGF ⁇ 1, TGF ⁇ 2, TIMP1, and ⁇ SMA than the HF- group. That is, a decrease in genes related to fibrosis was confirmed in the liver of the HF + group.
  • Fig. 20 shows a summary of the analysis item results.
  • an extract from a oyster containing a high concentration of 3,5-dihydroxy-4-methoxybenzyl alcohol was used in anti-obesity, anti-fatty liver, anti-inflammation, anti-fibrosis in a NASH model mouse. Excellent effects such as oxidization, anti-apoptosis, anti-oxidation, blood glucose level lowering, and plasma lipid lowering were confirmed.
  • FIG. 39 shows representative stained images of NC, HF + and HF ⁇ groups using both antibodies.
  • HF + stained images interspersed with both antibodies were observed.
  • HF-group infiltration of lymphocytes and macrophages into the liver was observed compared to the NC group.
  • HF + group almost no infiltration was observed in both blood cells compared to the HF- group.
  • FIG. 40 shows a graph in which the occupation ratio of both immunostained images performed in FIG. 39 is quantified using ImageJ.
  • the HF- group showed a statistically significant increase in staining images compared to the NC group, but the HF + group showed a statistically significant decrease compared to the HF- group. From these results, the anti-inflammatory action of the NASH model mouse by the aforementioned oyster extract was observed.
  • FIG. 41 shows a representative staining image and the number of staining in TUNEL assay.
  • the cell nucleus was stained. From the graph of the number of stained cells, the HF- group showed a statistically significant increase in the number of cells causing DNA fragmentation compared to the NC group, but the HF + group compared to the HF- group. A statistically significant decrease was observed. From these results, the anti-apoptotic action of NASH model mice was observed with the above-mentioned oyster extract.
  • FIG. 42 shows representative immunohistochemically stained images of NC, HF +, and HF + groups using anti-collagen antibodies, and a graph quantified using ImageJ.
  • HF-group a marked collagen staining image was seen around the venule.
  • HF- group an increase in the area where collagen accumulation was statistically significant was observed compared to the NC group, but in the HF + group, a statistically significant decrease was observed compared to the HF- group. Observed. From these results, the antifibrotic effect of NASH model mice was observed with the oyster extract.
  • FIG. 43 shows representative immunohistochemically stained images of NC, HF +, and HF + groups using both antibodies.
  • the stained image using the anti-8OHdG antibody showed a stained image in the cell nucleus, and the stained image using the anti-dityrosine antibody showed a stained image in the cytoplasm.
  • the HF-group an increase in the staining area was observed for both antibodies compared to the NC group.
  • the HF + group a decrease in the staining area was observed for both antibodies compared to the HF- group.
  • FIG. 44 shows a graph obtained by quantifying the occupation ratio of the immunostained image with the anti-8OHdG antibody and the anti-dityrosine antibody performed in FIG. 43 using ImageJ, and a graph of the amount of TBARS which is the oxidation state of lipid.
  • a statistically significant increase was observed in the HF-group compared to the NC group in both the occupancy ratio of the immunostained images with both antibodies and the amount of TBARS.
  • a statistically significant decrease was observed in the HF + group compared to the HF- group.
  • the transcription factor PPAR ⁇ ⁇ involved in fatty liver and insulin resistance and the receptor CD36 are shown. According to previous research, there are the following reports. 1. The main transcript of PPAR ⁇ in human fatty liver was CD36. 2. CD36 was deeply involved in insulin resistance and hyperinsulinemia in NAFLD patients.
  • observing the expression levels of PPAR ⁇ and CD36 is very important for observing fatty liver and insulin resistance.
  • FIG. 47 shows representative immunohistochemically stained images of NC, HF +, and HF + groups using both antibodies. Stained images using the anti- PPAR ⁇ antibody showed a stained image in the cell nucleus, and stained images using the anti-CD36 antibody showed a stained image in the cytoplasm.
  • an increase in the staining area was observed for both antibodies compared to the NC group.
  • a decrease in the staining area was observed for both antibodies compared to the HF- group.
  • FIG. 48 shows a graph in which the occupation ratio of the immunostained image performed in FIG. 47 is quantified using ImageJ.
  • the HF- group an increase in the occupation rate was observed compared with the NC group, but in the HF + group, a statistically significant decrease in the occupation rate was observed compared with the HF- group. From these results, a statistically significant decrease in the expression level of both proteins was observed in the liver of NASH model mice by the above-described oyster extract.
  • FIG. 49 shows gene expression levels of transcription factor PPAR ⁇ and receptor CD36 in the liver.
  • PPAR ⁇ and receptor CD36 were observed in the livers of NASH model mice by the oyster extract.
  • FIG. 50 shows a summary of the analysis item results in FIGS.

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  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

[Problem] The purpose of the present invention is to provide: a hepatoprotective agent that, by containing 3,5-dihydroxy-4-methoxybenzyl alcohol, has excellent hepatoprotective effects, including a hepatocyte protective effect, anti-apoptosis effect, anti-fatty liver effect, anti-NASH effect, anti-inflammatory effect, and anti-fibrosis effect; a glucose metabolism-improving agent; and an anti-obesity agent. [Solution] The present invention is characterized in that the active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol.

Description

肝臓保護剤、糖代謝改善剤及び抗肥満剤Liver protective agent, glucose metabolism improving agent and anti-obesity agent
　本発明は抗酸化剤である3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を含む肝臓保護剤、糖代謝改善剤及び抗肥満剤に関するものである。
  The present invention relates to a liver protective agent, a glucose metabolism improving agent and an anti-obesity agent containing 3,5-dihydroxy-4-methoxybenzyl alcohol, which is an antioxidant.
　活性酸素の生成は好気性の生活に起因し、脂質、タンパク質、核酸の酸化を生じ、細胞に障害を与えることが一般に知られている。 It is generally known that the generation of active oxygen is caused by aerobic life and causes oxidation of lipids, proteins and nucleic acids, and damages cells.
　通常、生体の酸化レベルは活性酸素産生系と抗酸化物質による消去系のバランスでほぼ一定に保たれているが、薬物、放射線、虚血などの様々な要因によりこのバランスが崩れ、活性酸素産生系へ傾くのが酸化ストレスといわれている。 Normally, the level of oxidation in the living body is almost constant by the balance between the active oxygen production system and the elimination system by antioxidants, but this balance is lost due to various factors such as drugs, radiation, and ischemia, and active oxygen production Leaning toward the system is said to be oxidative stress.
　この酸化ストレスの蓄積が、がん、動脈硬化性疾患、虚血/再灌流障害、慢性関節リウマチ、糖尿病、アルツハイマー病やパーキンソン病の神経障害などの様々な疾患や老化の一因であると考えられているのである。 This accumulation of oxidative stress is thought to contribute to various diseases and aging such as cancer, arteriosclerotic disease, ischemia / reperfusion injury, rheumatoid arthritis, diabetes, Alzheimer's disease and Parkinson's disease neuropathy It is.
　いわゆる抗酸化物質は構造から大きく二群に分類される。酵素性抗酸化物質としては、スーパーオキシドジスムターゼ(superoxidedismutase、SOD)、カタラーゼ(catalase、CAT)、グルタチオンペルオキシダーゼ(glutathioneperoxidase、GPx)、グルタチオンS-トランスフェラーゼ(glutathioneS-transferase、GST)、グルタチオンリダクターゼ(glutathionereductase)、ペルオキシレドキシン(peroxiredoxin、Prx)などが挙げられる。一方、非酵素性抗酸化物としては、アスコルビン酸(ascorbicacid)、α-トコフェロール(α-tocopherol)、グルタチオン(glutathione、GSH)、カロテノイド(carotenoids)、フラボノイド(flavonoids)、メタロチオネイン(metallothionein)などを含む。 So-called antioxidants are roughly classified into two groups based on their structure. Examples of enzymatic antioxidants include superoxide dismutase (superoxidedismutase, SOD), catalase (catalase, CAT), glutathione peroxidase (glutathioneperoxidase, GPx), glutathione S-transferase (glutathione S-transferase, GST), glutathione reductase (glutathionereductase), Examples include peroxiredoxin (Prx). On the other hand, non-enzymatic antioxidants include ascorbic acid, α-tocopherol, α-tocopherol, glutathione, GSH, carotenoids, flavonoids, metallothionein, etc. .
　ところで、カキ、たとえばマガキ(Crassostreagigas)はウグイスガイ目イタボガキ科に属する二枚貝で、その生息地は日本を初めとして東アジア全域に及んでいる。近年では、フランスやオーストラリアでもマガキが養殖されており、世界で最も食用に供さるカキとして名高い。 By the way, oysters, for example, oysters (Crassostreagigas) are bivalves belonging to the order of the squirrel crabs, and their habitat extends throughout Japan and other parts of East Asia. In recent years, oysters have been cultivated in France and Australia and are famous as the most edible oysters in the world.
　カキは、栄養価が高いことから古代より食用にされてきたが、前述したとおりグリコーゲンやタンパク質のほか、カルシウム、亜鉛、セレニウム、銅、マンガンなどのミネラルを多量に含むといわれている。 Oysters have been edible since ancient times due to their high nutritional value, but as mentioned above, they are said to contain a large amount of minerals such as calcium, zinc, selenium, copper, and manganese in addition to glycogen and protein.
　また、カキ由来の抗酸化物として報告されているのは、酵素性抗酸化物質としてSOD、CAT、GPx、及びPrx6があり、非酵素性抗酸化物質としてはメタロチオネイン、uncouplingprotein5(UCP5)、アスコルビン酸、α-トコフェロール、β-カロテンがあった。
  Moreover, SOD, CAT, GPx, and Prx6 are reported as antioxidants derived from oysters, and metallothionein, uncouplingprotein5 (UCP5), ascorbic acid are used as non-enzymatic antioxidants. , Α-tocopherol, and β-carotene.
特開２０１０－１９３７５６号公報JP 2010-193756 A
　しかして、本件発明の発明者らは、カキからの優れたいわゆる新規抗酸化物質を見出すことに成功し、さらにその化学構造を決定し、なおかつ前記抗酸化物質の化学合成を行うことにも成功し、そして、カキに由来しない、あるいはカキに由来する場合の双方での優れたいわゆる新規抗酸化剤及び抗酸化剤組成物の提供が行えることにも成功した。 Thus, the inventors of the present invention succeeded in finding an excellent so-called new antioxidant substance from oysters, further determining its chemical structure, and succeeding in chemical synthesis of the antioxidant substance. In addition, the present inventors have succeeded in providing so-called novel antioxidants and antioxidant compositions which are excellent both when they are not derived from oysters or when they are derived from oysters.
　さらに、ヒト低比重リポ蛋白（low-densitylipoproteins、LDL)の酸化実験と、肝臓の株化細胞の酸化実験における当該物質の抗酸化能をも確認でき、さらには、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分として、肝細胞保護作用、抗アポトーシス作用、抗脂肪肝作用、抗非アルコール性脂肪肝炎（NASH）作用、肝臓における抗炎症作用、肝臓における抗線維化作用を含む肝臓保護作用を確認でき、よって3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を含んで優れた肝細胞保護作用、抗アポトーシス作用、抗脂肪肝作用、抗NASH作用、抗炎症作用、抗線維化作用を含む肝臓保護作用を有する肝臓保護剤、糖代謝改善剤及び抗肥満剤の提供を目的とするものである。
  Furthermore, the antioxidant ability of the substance in human low-density lipoproteins (LDL) oxidation experiments and liver cell oxidization experiments can be confirmed. Furthermore, 3,5-dihydroxy-4- Using methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient, hepatocyte protection, anti-apoptosis, anti-fatty liver, anti-non-alcoholic steatohepatitis (NASH), anti-inflammatory in the liver , Liver protective action including anti-fibrotic action in the liver can be confirmed, and thus excellent hepatocyte protective action including 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol), It is intended to provide a liver protective agent having a liver protective action including an anti-apoptotic action, an anti-fatty liver action, an anti-NASH action, an anti-inflammatory action, and an anti-fibrotic action, a glucose metabolism improving agent, and an anti-obesity agent.
　本発明は、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
ことを特徴とし、
　または、
　カキ肉から抽出された抽出物に含有する3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
　ことを特徴とし、
　または、
　酢酸エチルを用いてカキ肉から3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)分画を回収し、該回収した3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
　ことを特徴とし、
　または、
　酢酸エチルとエタノールを用いてカキ肉から3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)分画を回収し、該回収した3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
　ことを特徴とし、
　または、
　抽出用溶液が貯留された抽出容器にカキ肉を収納してカキ肉抽出物入り溶液を採取し、採取されたカキ肉抽出物入り溶液に酢酸エチルを加えて、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)分画を回収し、該回収した3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
　ことを特徴とし、
　または、
　抽出用溶液が貯留された抽出容器にカキ肉を収納してカキ肉抽出物入り溶液を採取し、採取されたカキ肉抽出物入り溶液に酢酸エチルとエタノールを加えて、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)分画を回収し、該回収した3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
　ことを特徴とし、
　または、
　抽出用溶液が貯留された抽出容器にカキ肉を収納してカキ肉抽出物入り溶液を採取し、採取されたカキ肉抽出物入り溶液にエタノールを加えて、上澄み液と沈殿物とに分離し、前記分離された上澄み液を取り出すとともに、該上澄み液に酢酸エチルを加えて、酢酸エチル層と水層とに分離し、
　分離した酢酸エチル層の溶液を濃縮して3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)分画を回収し、該回収した3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
　ことを特徴とし、
　または、
　前記肝臓保護剤は、肝細胞保護作用を有する、
ことを特徴とし、
　または、
　前記肝臓保護剤は、抗アポトーシス作用を有する、
ことを特徴とし、
　または、
　前記肝臓保護剤は、抗脂肪肝作用を有する、
　ことを特徴とし、
　または、
　前記肝臓保護剤は、抗NASH作用を有する、
ことを特徴とし、
　または、
　前記肝臓保護剤は、肝臓における抗炎症作用を有する、
ことを特徴とし、
　または、
　前記肝臓保護剤は、肝臓における抗線維化作用を有する、
ことを特徴とし、
　または、
　前記糖代謝改善剤は、糖代謝改善作用を有する、
　ことを特徴とし、
　または、　　
　前記抗肥満剤は、抗肥満作用を有する、
　ことを特徴とし、
　または、
　前記肝臓保護剤は、肝臓におけるDNA、脂質、及びタンパク質における抗酸化作用を有する、
　ことを特徴とし、
　または、
　前記肝臓保護剤は、インスリン抵抗性改善作用を有する、
　ことを特徴とするものである。　　
  The present invention comprises 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
It is characterized by
Or
3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) contained in the extract extracted from oyster meat as an active ingredient,
It is characterized by
Or
A fraction of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was recovered from oyster meat using ethyl acetate, and the recovered 3,5-dihydroxy-4-methoxybenzyl alcohol was recovered. Alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
It is characterized by
Or
3,5-dihydroxy-4-methoxybenzyl alcohol fraction was recovered from oyster meat using ethyl acetate and ethanol, and the recovered 3,5-dihydroxy-4- Methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
It is characterized by
Or
The oyster meat is stored in the extraction container in which the extraction solution is stored, the solution containing the oyster meat extract is collected, ethyl acetate is added to the collected solution containing the oyster meat extract, and 3,5-dihydroxy-4-methoxybenzyl The alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) fraction is recovered, and the recovered 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is used as an active ingredient. ,
It is characterized by
Or
The oyster meat is stored in the extraction container in which the extraction solution is stored, and the solution containing the oyster meat extract is collected. Ethyl acetate and ethanol are added to the collected solution containing the oyster meat extract, and 3,5-dihydroxy-4- The fraction of methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is recovered, and the recovered 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is an active ingredient. And
It is characterized by
Or
The oyster meat is stored in the extraction container in which the extraction solution is stored, the solution containing the oyster meat extract is collected, ethanol is added to the collected solution containing the oyster meat extract, and the supernatant liquid and the precipitate are separated. The separated supernatant is taken out, and ethyl acetate is added to the supernatant to separate the ethyl acetate layer and the aqueous layer,
The separated ethyl acetate layer solution was concentrated to recover 3,5-dihydroxy-4-methoxybenzyl alcohol fraction, and the recovered 3,5-dihydroxy-4 -Methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
It is characterized by
Or
The hepatoprotective agent has a hepatocyte protective effect,
It is characterized by
Or
The hepatoprotective agent has an anti-apoptotic effect,
It is characterized by
Or
The hepatoprotective agent has an anti-fatty liver effect,
It is characterized by
Or
The hepatoprotective agent has an anti-NASH effect,
It is characterized by
Or
The hepatoprotectant has an anti-inflammatory effect in the liver,
It is characterized by
Or
The hepatoprotective agent has an antifibrotic effect in the liver,
It is characterized by
Or
The sugar metabolism improving agent has a sugar metabolism improving action,
It is characterized by
Or
The anti-obesity agent has an anti-obesity action,
It is characterized by
Or
The hepatoprotectant has an antioxidant action on DNA, lipids, and proteins in the liver,
It is characterized by
Or
The hepatoprotectant has an insulin resistance improving action,
It is characterized by this.
　本発明によれば、カキに由来しない、あるいはカキに由来する
　3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする肝細胞保護作用、抗アポトーシス作用、抗脂肪肝作用、抗NASH作用、抗炎症作用、抗線維化作用、糖代謝改善作用、抗肥満作用を有する肝臓保護剤、糖代謝改善剤及び抗肥満剤を提供できるとの優れた効果を奏する。
  According to the present invention, hepatocellular protective action, anti-antibacterial agent comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) not derived from oysters or derived from oysters as an active ingredient. Excellent ability to provide apoptotic action, anti-fatty liver action, anti-NASH action, anti-inflammatory action, anti-fibrosis action, glucose metabolism improving action, liver protective agent having anti-obesity action, sugar metabolism improving agent and anti-obesity agent There is an effect.
本発明の実験計画を説明する説明図である。It is explanatory drawing explaining the experiment plan of this invention. 本発明の分析項目を示す説明図である。It is explanatory drawing which shows the analysis item of this invention. 各マウスのHF+群とHF-群の摂餌量を示す説明図である。It is explanatory drawing which shows the food intake of HF + group and HF- group of each mouse | mouth. マウスの外観と肝臓の色調を分析した結果を示す説明図である。It is explanatory drawing which shows the result of having analyzed the external appearance of the mouse | mouth and the color tone of the liver. 実験開始１～２３週間のNC群、HF-群及びHF+群の各マウスにおける体重の変化を分析した結果を示す説明図である。FIG. 6 is an explanatory diagram showing the results of analyzing changes in body weight in mice of NC group, HF− group, and HF + group from 1 to 23 weeks after the start of the experiment. NC群、HF-群及びHF+群の各マウスの体重と肝重量を分析した結果を示す説明図である。It is explanatory drawing which shows the result of having analyzed the body weight and liver weight of each mouse | mouth of NC group, HF- group, and HF + group. 病理学的観察におけるスコアの基準を説明する説明図である。It is explanatory drawing explaining the standard of the score in pathological observation. 各判定の正常細胞及び病的細胞の写真を示す説明図である。It is explanatory drawing which shows the photograph of the normal cell and pathological cell of each determination. NC群、HF-群及びHF+群の各マウスの肝臓の病理学的観察の結果を示す説明図である。It is explanatory drawing which shows the result of the pathological observation of the liver of each mouse | mouth of NC group, HF- group, and HF + group. NC群、HF-群及びHF+群の各マウスの肝臓の各脂質の濃度と脂質の過酸化度(TBARS)を分析した結果を示す説明図である。It is explanatory drawing which shows the result of having analyzed the density | concentration of each lipid of the liver of each mouse | mouth of NC group, HF- group, and HF + group, and the lipid peroxidation degree (TBARS). NC群、HF-群及びHF+群の各マウス血漿中のAST活性、ALT活性について分析した結果を示す説明図である。It is explanatory drawing which shows the result of having analyzed about AST activity and ALT activity in each mouse | mouth plasma of NC group, HF- group, and HF + group. NC群、HF-群及びHF+群の各マウス血漿中のグルコース、インスリン濃度及びインスリン抵抗性指数について分析した結果を示す説明図である。It is explanatory drawing which shows the result analyzed about the glucose in each mouse | mouth plasma of an NC group, HF- group, and HF + group, an insulin concentration, and an insulin resistance index. NC群、HF-群及びHF+群の各マウスの血漿の各脂質の濃度を分析した結果を示す説明図である。It is explanatory drawing which shows the result of having analyzed the density | concentration of each lipid of the plasma of each mouse | mouth of NC group, HF- group, and HF + group. 肝臓における炎症反応に関する遺伝子について分析した結果を示す説明図である。It is explanatory drawing which shows the result of having analyzed about the gene regarding the inflammatory reaction in a liver. 肝臓における炎症反応に関する遺伝子について説明する説明図である。It is explanatory drawing explaining the gene regarding the inflammatory reaction in a liver. 肝臓におけるアポトーシスに関する遺伝子について分析した結果を示す説明図である。It is explanatory drawing which shows the result of having analyzed about the gene regarding the apoptosis in a liver. 肝臓におけるアポトーシスに関する遺伝子について説明する説明図である。It is explanatory drawing explaining the gene regarding the apoptosis in a liver. 肝臓における線維化に関する遺伝子について分析した結果を示す説明図である。It is explanatory drawing which shows the result of having analyzed about the gene regarding the fibrosis in a liver. 肝臓における線維化に関する遺伝子について説明する説明図である。It is explanatory drawing explaining the gene regarding the fibrosis in a liver. 分析項目の結果のまとめを示す説明図である。It is explanatory drawing which shows the summary of the result of an analysis item. 本発明の実験の結語を示す説明図である。It is explanatory drawing which shows the conclusion of the experiment of this invention. 本発明で効果の得られる作用について説明する説明図である。It is explanatory drawing explaining the effect | action from which an effect is acquired by this invention. 本発明の概略構成を示す概略構成説明図（１）である。BRIEF DESCRIPTION OF THE DRAWINGS It is schematic structure explanatory drawing (1) which shows schematic structure of this invention. 本発明の概略構成を示す概略構成説明図（２）である。It is schematic structure explanatory drawing (2) which shows schematic structure of this invention. 本発明のフローチャートを説明する説明図である。It is explanatory drawing explaining the flowchart of this invention. 3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)抽出のための有機溶媒の極性を段階的に高めていく状態を説明する説明図である。It is explanatory drawing explaining the state which raises the polarity of the organic solvent for 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl-4-alcohol) extraction stepwise. 各抽出物の抗酸化活性試験の結果を示す説明図である。It is explanatory drawing which shows the result of the antioxidant activity test of each extract. シリカオープンカラムによる抽出を説明する説明図である。It is explanatory drawing explaining extraction by a silica open column. 酢酸エチル分画抽出を説明する説明図である。It is explanatory drawing explaining ethyl acetate fraction extraction. 本発明により抽出された3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)の構造を説明する説明図である。It is explanatory drawing explaining the structure of 3, 5- dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) extracted by this invention. ORAC法の測定原理を説明する説明図である。It is explanatory drawing explaining the measurement principle of ORAC method. 本発明による3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)の抗酸化能を説明する説明図である。It is explanatory drawing explaining the antioxidant ability of 3, 5- dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) by this invention. LDLの酸化度をTBARS法により定量した状態を説明する説明図である。It is explanatory drawing explaining the state which quantified the oxidation degree of LDL by TBARS method. diphenyl-1-pyrenlphosphine（DPPP）の蛍光発色の原理を説明する説明図である。It is explanatory drawing explaining the principle of the fluorescence coloring of diphenyl-1-pyrenlphosphine (DPPP). 本発明における3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を添加した細胞の酸化抑制状態を説明する説明図である。It is explanatory drawing explaining the oxidation suppression state of the cell which added 3, 5- dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) in this invention. 本発明による3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)の構造解析を説明する説明図（１）である。It is explanatory drawing (1) explaining the structural analysis of 3, 5- dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) by this invention. 本発明による3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)の構造解析を説明する説明図（２）である。It is explanatory drawing (2) explaining the structural analysis of 3, 5- dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) by this invention. 3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)の化学合成を説明する説明図である。It is explanatory drawing explaining the chemical synthesis of 3, 5- dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol). 肝臓の炎症反応の特徴であるリンパ球とマクロファージの浸潤を抗-CD3抗体(リンパ球に特異的な抗体)と抗-F4/80 (マクロファージに特異的な抗体)を用いたNC、HF+、HF-群の代表的な染色像を説明する説明図である。NC, HF +, HF using anti-CD3 antibody (antibody specific for lymphocytes) and anti-F4 / 80 (antibody specific for macrophages) to infiltrate lymphocytes and macrophages, which are characteristic of liver inflammatory response -It is explanatory drawing explaining the typical dyeing | staining image of a group. 両免疫染色像の占有率をImageJを用いて定量化したグラフを説明する説明図である。It is explanatory drawing explaining the graph which quantified the occupation rate of both the immuno-staining images using ImageJ. TUNEL assayにおける代表的な染色像と染色数のグラフを説明する説明図である。It is explanatory drawing explaining the graph of the typical dyeing | staining image and dyeing | staining number in TUNEL assay. 抗-コラーゲン抗体を用いたNC、HF+、HF+群の代表的な免疫組織化学染色像、及びImageJを用いて定量化したグラフを説明する説明図である。It is explanatory drawing explaining the graph quantified using the typical immunohistochemical dyeing | staining image of NC, HF +, HF + group using an anti- collagen antibody, and ImageJ. 肝臓における酸化の状態をDNAの酸化マーカーである8OHｄGの抗体を用いて、タンパク質マーカーであるdityrosineの抗体を用いたNC、HF+、HF+群の代表的な免疫組織化学染色像を説明する説明図である。In the explanatory diagram explaining the typical immunohistochemical staining image of the NC, HF +, HF + group using the antibody of dityrosine, the protein marker, using the 8OHdG antibody, which is the DNA oxidation marker, the oxidation state in the liver is there. 抗-8OHｄG抗体と抗-dityrosine抗体での免疫染色像の占有率をImageJを用いて定量したグラフ、並びに脂質の酸化状態であるTBARS量のグラフを説明する説明図である。It is explanatory drawing explaining the graph which quantified the occupation rate of the immuno-staining image with an anti-8 OHdG antibody and an anti- dityrosine antibody using ImageJ, and the graph of the amount of TBARS which is the oxidation state of a lipid. マガキの抽出物によりNASHモデルマウスの肝臓におけるDNA、脂質、及びタンパク質における抗酸化性を説明する説明図である。It is explanatory drawing explaining the antioxidant property in DNA, a lipid, and protein in the liver of a NASH model mouse by the extract of a oyster. 脂肪肝やインスリン抵抗性に関わる転写因子PPARγと受容体CD36を説明する説明図である。It is explanatory drawing explaining the transcription factor PPARγ and receptor CD36 which are related to fatty liver and insulin resistance. 肝臓での転写因子PPARγを抗- PPARγ抗体、受容体CD36を抗- CD36抗体を用いたNC、HF+、HF+群の代表的な免疫組織化学染色像を説明する説明図である。It is explanatory drawing explaining the typical immunohistochemical dyeing | staining image of NC, HF +, and HF + group using the transcription factor PPARγ in the liver using anti- PPARγ antibody and the receptor CD36 using anti- CD36 antibody. 免疫染色像の占有率をImageJを用いて定量化したグラフを説明する説明図である。It is explanatory drawing explaining the graph which quantified the occupation rate of the immuno-staining image using ImageJ. 肝臓での転写因子PPARγ、受容体CD36の遺伝子発現量を説明する説明図である。It is explanatory drawing explaining the gene expression level of transcription factor PPARγ and receptor CD36 in the liver. 図３９から図４９における分析項目の結果のまとめを説明する説明図である。It is explanatory drawing explaining the summary of the result of the analysis item in FIGS. 39-49.
　以下、本発明を図に示す一実施例に基づいて説明する。 Hereinafter, the present invention will be described based on an embodiment shown in the drawings.
　図２３、図２４において、符号１は、抽出容器であり、該抽出容器１内には、カキ肉から抽出物を抽出するための抽出用溶液２が貯留される。そして、該抽出用溶液２が貯留されている抽出容器１内に生ガキ肉３を収納し、カキ肉の各種有効成分を含有する抽出物を抽出する工程が行われる。 23 and 24, reference numeral 1 denotes an extraction container, in which an extraction solution 2 for extracting an extract from oyster meat is stored. And the raw oyster meat 3 is accommodated in the extraction container 1 in which this extraction solution 2 is stored, and the process of extracting the extract containing various active ingredients of oyster meat is performed.
　ところで、従来では、カキ肉抽出物抽出時に、抽出容器１内のカキ肉３が収納された抽出用溶液２を攪拌し、抽出をより効率化することが従来行われていたことがあったが、カキ肉３自体を痛めることにもなり、この抽出工程時点での攪拌作業は行わない方が好ましい。 By the way, conventionally, when extracting the oyster meat extract, the extraction solution 2 containing the oyster meat 3 in the extraction container 1 was agitated to make the extraction more efficient. The oyster meat 3 itself may be damaged, and it is preferable not to perform the stirring operation at the time of the extraction process.
　前述のようにしてカキ肉抽出物が抽出された抽出用溶液２を次は濃縮工程によって濃縮されるものとなる。 The extraction solution 2 from which the oyster meat extract has been extracted as described above is then concentrated in the concentration step.
　次に、この濃縮液６に、エタノール溶液４を加え、70%程度のエタノール濃度の溶液とする。その後、攪拌すると共に、沈殿物７と上澄み液８とに分離する。 Next, the ethanol solution 4 is added to the concentrated solution 6 to obtain a solution having an ethanol concentration of about 70%. Thereafter, the mixture is stirred and separated into a precipitate 7 and a supernatant 8.
　そして、図２３から理解されるように、沈殿物７は乾燥させ、打錠し、最終的に健康食品などに供される。 And as can be understood from FIG. 23, the precipitate 7 is dried, tableted, and finally used for health food.
　ところで、従来では前記上澄み液８は、何らカキ肉抽出物の有効成分が入っていない、あるいは入っていてもきわめて微量であるとして廃棄していたことがある。しかし、その後、実験や研究の結果、この上澄み液８内にもカキ肉抽出物に関する多くの有効成分が存在していることが判明し、現在ではこの上澄み液８も廃棄することなく利用している。 By the way, conventionally, the supernatant liquid 8 may be discarded because it does not contain any active ingredient of oyster meat extract or is contained in a very small amount. However, after that, as a result of experiments and researches, it became clear that many active ingredients relating to the oyster meat extract were also present in this supernatant liquid 8, and now this supernatant liquid 8 is used without being discarded. Yes.
　近年では、この上澄み液８を再度濃縮するとともに、その濃縮液を最終的に乾燥させる。そして、その乾燥物は、完全な固形物状にはならないが、ペースト状には形成することができ、もってペースト状の健康食品とするなどして製造している。そして、このペースト状の健康食品は、需要者側において白湯などで溶いて飲料用健康食品に供されるのである。 In recent years, the supernatant 8 is concentrated again and the concentrated solution is finally dried. The dried product does not become a complete solid, but can be formed into a paste, and is manufactured as a paste-like health food. And this paste-form health food is melt | dissolved with a white hot water etc. in the consumer side, and is provided to the health food for drinks.
　まず、本実施例では、前記の上澄み液８を使用して後述する3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)入りのカキ肉抽出物を回収するものである。 First, in this example, oyster meat extract containing 3,5-dihydroxy-4-methoxybenzyl alcohol, which will be described later, is recovered using the supernatant 8 described above. Is.
　すなわち、前記のごとく沈殿物７と上澄み液８に分離した後、該上澄み液８につき、まず、エバポレータなどで前記上澄み液８のエタノール分を除去し、約半分の量になるまで濃縮する。 That is, after separating into the precipitate 7 and the supernatant 8 as described above, first, the ethanol content of the supernatant 8 is removed from the supernatant 8 with an evaporator or the like, and concentrated to about a half amount.
　たとえば40mL分の上澄み液８を濃縮して20mLの上澄み液８の濃縮液９を確保するがごときである。 For example, a 40 mL portion of the supernatant 8 is concentrated to ensure a 20 mL supernatant 8 concentrate 9.
　次いで、その20mLの濃縮液を約５倍になるよう希釈して希釈液１０を生成する。たとえば100mLの希釈液１０の量にするがごときである。このような工程を経るのはなるべく不純物を除去するためである。 Next, the diluted solution 10 is produced by diluting the 20 mL concentrated solution to about 5 times. For example, the amount of the diluted solution 10 is 100 mL. This process is performed in order to remove impurities as much as possible.
　その後、たとえばこの100mLの希釈液１０の溶液に、たとえば酢酸エチル５を200mL程度投入する。そして、その後攪拌するなどして、あるいは分離器を使用して水層１０ａと酢酸エチル層１１とに分離させる。すると、時間の経過と共に、この混合溶液は、水層１０ａ、そして酢酸エチル層１１とに分離して形成されるものとなる。 Then, for example, about 200 mL of ethyl acetate 5 is added to the 100 mL of the diluted solution 10. Then, the aqueous layer 10a and the ethyl acetate layer 11 are separated by stirring or using a separator. Then, with the passage of time, this mixed solution is formed separately into the aqueous layer 10a and the ethyl acetate layer 11.
　すると、この酢酸エチル層１１内に後述する3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)が存在していることが確認できた。 Then, it was confirmed that 3,5-dihydroxy-4-methoxybenzyl alcohol described later was present in the ethyl acetate layer 11.
　ここで、その確認できた3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)の量であるが、具体的には、約2L分収集した酢酸エチル層１１から約3mgの3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)があることが確認できた。 Here, the amount of 3,5-dihydroxy-4-methoxybenzyl alcohol (3, 5-dihydroxy-4-methoxybenzyl alcohol) that could be confirmed, specifically, the ethyl acetate layer 11 collected for about 2 L. It was confirmed that there was about 3 mg of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol).
　次に、前記3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)がどの様な工程でカキ肉抽出物から分離精製でき、もってカキ肉抽出物内での存在が確認できたのか、また3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)はどのような構造から構成されているのか、さらには3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)の抗酸化作用がどの様に確認できたのかなどを以下に説明する。 Next, the 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl キ alcohol) can be separated and purified from the oyster extract in any process, It was confirmed that it was present, what structure is 3,5-dihydroxy-4-methoxybenzyl alcohol, and moreover, 3,5-dihydroxy The following describes how the antioxidant action of -4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzylholalcohol) was confirmed.
　まず、図２５に示すフローチャートに従って説明する。 First, a description will be given according to the flowchart shown in FIG.
　たとえば、エタノール溶液４を含んだ抽出用溶液２内にカキ肉３を投入してカキ肉有効成分抽出物の抽出を行なう（ステップ１００、ステップ１０２）。 For example, the oyster meat 3 is put into the extraction solution 2 containing the ethanol solution 4 to extract the oyster meat active ingredient extract (step 100, step 102).
　抽出後はその抽出液を濃縮する（ステップ１０４）。そして、該濃縮液６にたとえば、エタノール溶液４を加え、70%程度のエタノール濃度の溶液とする（ステップ１０６）。その後、攪拌し、沈殿物７と上澄み液８とに分離する（ステップ１０８）。 After extraction, the extract is concentrated (step 104). Then, for example, the ethanol solution 4 is added to the concentrated solution 6 to obtain a solution having an ethanol concentration of about 70% (step 106). Then, it stirs and isolate | separates into the precipitate 7 and the supernatant liquid 8 (step 108).
　そして、前記上澄み液８を用い、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)抽出のための酢酸エチルを用いた抽出作業を行う。 Then, using the supernatant 8, an extraction operation using ethyl acetate for extraction of 3,5-dihydroxy-4-methoxybenzyl alcohol is performed.
　図２５から理解されるように、前記エタノール分をなくし（ステップ１１０）、かつ約５倍に希釈した上澄み液８におのおのヘキサンからクロロホルム、酢酸エチル、そしてブタノールを投入し、おのおのの分画を生成する。 As can be seen from FIG. 25, the ethanol content is eliminated (step 110), and chloroform, ethyl acetate, and butanol are added to hexane from each hexane into the supernatant 8 diluted approximately 5 times to produce each fraction. To do.
　例えば、ロータリーエバポレーターなどで100mLまで濃縮し、該濃縮液20mLに例えば蒸留水80mLを加えて分液ロートに移し、ヘキサン抽出を行う。 For example, concentrate to 100 mL with a rotary evaporator or the like, add 80 mL of distilled water to 20 mL of the concentrated solution, transfer to a separatory funnel, and perform hexane extraction.
　ヘキサン層(200mL)を除去後に、水層からクロロホルム200mL、酢酸エチル200mL、ブタノール200mLの順で段階的に抽出する。 After removing the hexane layer (200 mL), the aqueous layer is extracted stepwise in the order of 200 mL chloroform, 200 mL ethyl acetate, and 200 mL butanol.
　すなわち、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)抽出のための有機溶媒の極性を段階的に高めていってそれらをそれぞれ投入した分画を生成し、おのおのの分画に3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)が抽出されているかを確認する（図２６参照）。 That is, the polarity of organic solvents for 3,5-dihydroxy-4-methoxybenzylcoalcohol extraction is increased step by step, and fractions are added to each of them. Then, it is confirmed whether 3,5-dihydroxy-4-methoxybenzyl alcohol is extracted from each fraction (see FIG. 26).
　次いで、前記それぞれの有機溶媒を投入した分画をたとえばエバポレータで濃縮した後、Thin-Layer-Chromatography（以下、TLCと称する。TLC:薄層クロマトグラフィー）により観察すると共に、いわゆるORAC法（OxygenRadicalAbsorbanceCapacity法）による抗酸化力の検索を行うのである。 Next, the fractions charged with the respective organic solvents are concentrated by, for example, an evaporator, and then observed by thin-layer-chromatography (hereinafter referred to as TLC; TLC: thin-layer chromatography). ) To search for antioxidant power.
　すると、その結果、TLC像では、ヘキサンからクロロホルム、酢酸エチル、そしてブタノールにかけて極性の低いものから高いものへと溶出されていくことが確認できた。 As a result, in the TLC image, it was confirmed that elution was performed from hexane to chloroform, ethyl acetate, and butanol from low polarity to high polarity.
　また、ORAC法により酢酸エチル分画においてプラトーの部分が観察されて、当該酢酸エチル分画に高い抗酸化能が認められ、よって、この酢酸エチル分画に3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4- methoxybenzyl alcohol)が存在していると判断されるのである（図２７参照、ステップ１１２）。 In addition, a plateau portion was observed in the ethyl acetate fraction by the ORAC method, and a high antioxidant capacity was observed in the ethyl acetate fraction. Therefore, 3,5-dihydroxy-4-methoxybenzyl was present in the ethyl acetate fraction. It is determined that alcohol (3,5-dihydroxy-4- methoxybenzyl alcohol) is present (see FIG. 27, step 112).
　次いで、この酢酸エチル分画をエバポレータによりやはり濃縮した後、いわゆるシリカオープンカラムによる抽出を行い（図２８、ステップ１１２６）、酢酸エチル：クロロホルムが３：２の割合での抽出分画を選択し（図２９参照）、最終的にその分画をHPLC（逆相カラム）によって、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-
methoxybenzyl alcohol)を分離精製することができたのである（ステップ１１６）。 Next, the ethyl acetate fraction was concentrated using an evaporator and then extracted with a so-called silica open column (FIG. 28, step 1126), and the ethyl acetate / chloroform extraction fraction at a ratio of 3: 2 was selected ( 29), finally, the fraction was analyzed by HPLC (reverse phase column) to 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-
It was possible to separate and purify methoxybenzyl alcohol) (step 116).
　このように、カキ肉抽出物を抽出した上澄み液８から3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を分離精製することができた。 Thus, it was possible to separate and purify 3,5-dihydroxy-4-methoxybenzyl alcohol from the supernatant 8 from which the oyster meat extract was extracted.
　なお、以下の操作によっても3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を分離精製することができる。 Incidentally, 3,5-dihydroxy-4-methoxybenzyl alcohol can also be separated and purified by the following operation.
　まず、0.075mol/Lリン酸緩衝液2.3、5mL、6.3x10-7mol/LFluorescein(蛍光プローブ)0.3mL、7％(w/v)methylatedβ-cyclodextrin(Wako)の混合溶液に溶解したトロロックス(Wako)または被験試料0.05mLを37℃で10分間加温する。 First, Trolox (0.075 mol / L phosphate buffer 2.3, 5 mL, 6.3 x 10 -7 mol / L Fluorescein (fluorescent probe) 0.3 mL, 7% (w / v) methylated β-cyclodextrin (Wako) mixed solution) Wako) or 0.05 mL of the test sample is heated at 37 ° C. for 10 minutes.
　予め37℃に加温した1.28x10-1mol/L2、2’-azobis(2-amidinopropane)dihydrochloride(AAPH、Wako)0.3mLを加え、例えばスターラ―で撹拌しながら、分光蛍光光度計(FP-6500、JASCO、東京)で10秒おきに5,000秒まで蛍光強度(励起波長493nm、蛍光波長515nm)を測定する。 Add 1.28x10 -1 mol / L2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH, Wako) 0.3 mL, pre-warmed to 37 ° C, and stir with a stirrer, for example, with a spectrofluorometer (FP- Measure fluorescence intensity (excitation wavelength 493 nm, fluorescence wavelength 515 nm) every 10 seconds up to 5,000 seconds at 6500, JASCO, Tokyo).
　抗酸化活性は測定開始時点の蛍光測定値（例えば図２７中の縦軸）が維持される時間（同横軸）の長さで示され、その時間が長いほど抗酸化活性が強いことを意味するものである。 The antioxidant activity is indicated by the length of time (horizontal axis) during which the fluorescence measurement value at the start of measurement (for example, the vertical axis in FIG. 27) is maintained, and the longer the time, the stronger the antioxidant activity. To do.
　すると、やはり前記４種類の抽出画分の中では酢酸エチル抽出画分に抗酸化活性が確認された。 Then, the antioxidant activity was confirmed in the ethyl acetate extract fraction among the four kinds of extract fractions.
　次いで、抗酸化活性が示された酢酸エチル抽出物について順相のシリカゲル薄層分取クロマトグラフィーを行う。シリカゲル薄層プレート（200×200mm、厚さ0.5mm、Merck、Darmstadt）を用い、移動相として酢酸エチル-クロロホルム(2:1、v/v)を用いた。展開後のプレートに紫外線ランプ（254nm）を照射し、紫外線吸収性の11画分を得た。各画分の試料をゲル担体とともに分離し、例えばメタノールで溶出後に抗酸化活性を測定すると、低極性側から6番目の画分に抗酸化活性が観察された。 Subsequently, normal-phase silica gel thin layer preparative chromatography is performed on the ethyl acetate extract showing the antioxidant activity. Silica gel thin layer plates (200 × 200 mm, thickness 0.5 mm, Merck, Darmstadt) were used, and ethyl acetate-chloroform (2: 1, v / v) was used as the mobile phase. The developed plate was irradiated with an ultraviolet lamp (254 nm) to obtain 11 fractions absorbing ultraviolet rays. A sample of each fraction was separated together with a gel carrier, and when the antioxidant activity was measured after elution with methanol, for example, the antioxidant activity was observed in the sixth fraction from the low polarity side.
　さらに、前記薄層クロマトグラフィーで抗酸化活性を示した画分を高速液体クロマトグラフィー（HPLC）で精製する。HPLCシステム（ポンプ：L-2130、UV検出器：L-2420、HITACHI、東京）、逆相カラム(APCELLPACC18、250×4.6mmI.D.、SHISEIDO、東京)、及び移動相5%アセトニトリル水溶液（流速1.0mL/min）を使用して室温で分離した。 Further, the fraction showing the antioxidant activity by the thin layer chromatography is purified by high performance liquid chromatography (HPLC). HPLC system (pump: L-2130, UV detector: L-2420, HITACHI, Tokyo), reverse phase column (APCELLPACC18, 250 x 4.6 mm I.D., SHISEIDO, Tokyo), mobile phase 5% acetonitrile aqueous solution (flow rate) 1.0 mL / min) at room temperature.
　しかして、この操作によっても原料の160mLエタノール抽出液から最終的に3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4- methoxybenzyl alcohol)3.0mgが得られるものとなった。 However, even by this operation, 3.0 mg of 3,5-dihydroxy-4-methoxybenzyl alcohol was finally obtained from the raw 160 mL ethanol extract.
　ところで、前記3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4- methoxybenzyl alcohol)の存在は、紫外線吸収スペトル（V-530、JASCO）、核磁気共鳴スペクトル（NMR：AMX-500、Bruker、Karlsruhe）、マススペクトル（JMS-T100CS、JEOL、東京）を測定して、構造解析を行い(図３６、図３７)。その結果、前記の分離精製物の構造が、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4- methoxybenzyl alcohol)と推定されるのである(図３７)。 By the way, the presence of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4- methoxybenzyl alcohol) is caused by ultraviolet absorption spectrum (V-530, JASCO), nuclear magnetic resonance spectrum (NMR: AMX-). 500, Bruker, Karlsruhe) and mass spectrum (JMS-T100CS, JEOL, Tokyo) were measured and structural analysis was performed (FIGS. 36 and 37). As a result, the structure of the separated and purified product is presumed to be 3,5-dihydroxy-4-methoxybenzyl alcohol (FIG. 37).
　条件
UV（EtOH）、λmax270nm；1H-NMR(500MHz、Acetone-d6)δ・H:7.82(2H、br.s、aromatic-OH)、6.40(2H、s、H-2、6)、4.42(2H、s、H-1’)、3.94(1H、br.s、-OH)、3.79(3H、s、-OMe)；13C-NMR(125MHz、Acetone-d6)δC:151.1(C-3、5)、139.4(C-1)、13、5.1(C-4)、106.5(C-2、6)、64.5(C-1’)、60.6(-OMe)；ESI-TOFMS、m/z153.05451[M-OH]+(calc.forC8H8O3、153.05517)、171.06911[M+H]+(calc.forC8H11O4、171.06573)。 conditions
UV (EtOH), λ max 270 nm; 1 H-NMR (500 MHz, Acetone-d 6 ) δ · H : 7.82 (2H, br.s, aromatic-OH), 6.40 (2H, s, H-2, 6) , 4.42 (2H, s, H-1 ′), 3.94 (1H, br.s, —OH), 3.79 (3H, s, —OMe); 13 C-NMR (125 MHz, Acetone-d 6 ) δ C : 151.1 (C-3, 5), 139.4 (C-1), 13, 5.1 (C-4), 106.5 (C-2, 6), 64.5 (C-1 '), 60.6 (-OMe); ESI- TOFMS, m / z 153.05451 [M-OH] + (calc.forC 8 H 8 O 3 , 153.505517), 171.006911 [M + H] + (calc.forC 8 H 11 O 4 , 171.006573).
　ここで、分離精製された3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)の性状を説明すると、その性状は黄淡色の粉末で、脂溶性及び水溶性を示している。 Here, the properties of the separated and purified 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) will be explained. Its properties are pale yellow powder, fat-soluble and water-soluble. Is shown.
　また、当該3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)は、図３０に示すようなフェノール性化合物であることが確認された。 Further, it was confirmed that the 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is a phenolic compound as shown in FIG.
　なお、ここで、食品の抗酸化物質の測定法については、これまでに様々報告されてきているが、どれも一長一短があり、統一または公定法化(分析値の妥当性確認)された方法はなかった。しかしながら、米国では、すでにORAC値を表記したサプルメントや飲料が上市されており、世界標準となりつつある。 Here, various methods for measuring antioxidants in foods have been reported so far, but all have their merits and demerits, and the standardized or official method (confirmation of the validity of analytical values) There wasn't. However, in the United States, supplements and beverages that express ORAC values are already on the market and are becoming the global standard.
　よって、本実施例では、ORAC法により抗酸化力を測定することとしている。 Therefore, in this embodiment, the antioxidant power is measured by the ORAC method.
　ところで、日本ではすでにORAC法の公定法化の研究を行う研究会(AntioxidantUnit研究会)が出来ている。ORAC法の利点としては水溶性、脂溶性のどちらのサンプルも測定でき、前述したどの有機溶媒分画も測定できることがあげられる。 By the way, in Japan, there is already a study group (AntioxidantUnit Study Group) that conducts research on the formalization of the ORAC method. The advantage of the ORAC method is that both water-soluble and fat-soluble samples can be measured, and any of the organic solvent fractions described above can be measured.
　また一回の測定で抗酸化作用の持続時間とその力価を合わせて評価でき、実験操作が容易であるなどから本実施例での測定に有利であったと考える。 Also, the duration of the antioxidant action and its titer can be evaluated together with a single measurement, and the experimental operation is easy, so it is considered advantageous for the measurement in this example.
　ここで、ORAC法の測定原理について若干説明する。まず、一定の活性酸素種を発生させ、それによって分解される蛍光強度を測定し、経時的に減少する蛍光強度の曲線を描いた場合、この反応系に抗酸化物質が共存すると蛍光物質の蛍光強度の減少速度が遅延する。よって、この原理により抗酸化物質の存在が確認できるものとなるのである（図３１参照）。 Here, the measurement principle of the ORAC method will be explained briefly. First, when a certain reactive oxygen species is generated, the fluorescence intensity decomposed thereby is measured, and a fluorescence intensity curve that decreases with time is drawn, if an antioxidant coexists in this reaction system, the fluorescence of the fluorescent substance The rate of decrease in intensity is delayed. Therefore, the existence of an antioxidant substance can be confirmed by this principle (see FIG. 31).
　しかして、本発明における3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を前記ORAC法によりその抗酸化能を観察したところ、いわゆる標準物質（Trolox）と同じように延滞期が存在し、強い抗酸化活性が観察できたのである（図３２参照）。 Then, when the antioxidant ability of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) in the present invention was observed by the ORAC method, the so-called standard substance (Trolox) and Similarly, there was a postponement period and strong antioxidant activity could be observed (see FIG. 32).
　本実施例では、前述した上澄み液８から探査すべく、ORAC法を用い、酢酸エチル分画において高い抗酸化力を有する3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4- methoxybenzyl alcohol)を発見できたのである。 In this example, in order to search from the supernatant 8 described above, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4) having high antioxidant power in the ethyl acetate fraction was used using the ORAC method. -Methoxybenzyl alcohol) was discovered.
　次に、培養肝細胞の酸化実験と低密度リポタンパク質（LDL）の酸化実験の両者において、当該3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4- methoxybenzyl alcohol)の抗酸化活性を明らかにすることとする。 Next, in both cultured hepatocyte oxidation experiments and low-density lipoprotein (LDL) oxidation experiments, the 3,5-dihydroxy-4- methoxybenzyl alcohol was tested against The oxidation activity will be clarified.
　（当該物質の正常ヒトLDLの金属酸化に対する抗酸化活性について）
硫酸銅により正常ヒトLDLを酸化する際に、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-
methoxybenzyl alcohol)を添加し、LDLの酸化度をTBARS法により定量した(図３３参照)。すると、当該物質の180μM添加時にはControl(0μM)と同様に、lag-timeは観察されなかった。Lag-timeとは曲線の立ち上がりが見られない時間を指し、この時間が長くなるほど抗酸化活性があると判断される。しかし、270μM添加時には２時間、360μMでは４時間、450μM及び540μMでは５時間と、用量依存性にlag-timeは延長し、当該3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4- methoxybenzyl alcohol)によるLDLの金属酸化の抑制が確認されたのである。 (About the antioxidant activity of the substance against metal oxidation of normal human LDL)
When normal human LDL is oxidized by copper sulfate, 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-
Methoxybenzyl alcohol) was added, and the degree of oxidation of LDL was quantified by the TBARS method (see FIG. 33). Then, when 180 μM of the substance was added, lag-time was not observed as in Control (0 μM). Lag-time refers to the time when no rise of the curve is seen, and the longer this time is, the more it is judged that there is antioxidant activity. However, when 270 μM is added, the lag-time is prolonged in a dose-dependent manner, 2 hours for 360 μM, 4 hours for 360 μM and 5 hours for 450 μM and 540 μM, and the 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy -4-methoxybenzyl alcohol) was confirmed to suppress the metal oxidation of LDL.
　（肝細胞培養系を用いた抗酸化能の観察について）
diphenyl-1-pyrenlphosphine（DPPP）はそれ自体、蛍光を発生しないが、酸化されると蛍光を発生する。ここで、diphenyl-1-pyrenlphosphine（DPPP）の蛍光発色の原理を図３４に示す。この蛍光色素を用い、肝臓の株化細胞(C3A)の酸化度を観察したものである。 (Observation of antioxidant capacity using hepatocyte culture system)
Diphenyl-1-pyrenlphosphine (DPPP) itself does not fluoresce, but fluoresces when oxidized. Here, the principle of fluorescence development of diphenyl-1-pyrenlphosphine (DPPP) is shown in FIG. This fluorescent dye was used to observe the degree of oxidation of the liver cell line (C3A).
　そして、本発明における3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を添加した細胞を５日間培養し、その後、DPPPで標識した細胞を2、2’-azobis(2-methylpropionamidine)dihydrochloideにより酸化し、各細胞のDPPPの蛍光強度を測定した。その結果、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4- methoxybenzyl alcohol)を細胞に添加しなかったものと比較し、添加した細胞は濃度依存的に蛍光強度が低く、当該抗酸化物質3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4- methoxybenzyl alcohol)による酸化抑制が観察されたのである(図３５参照)。 Then, the cells to which 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) in the present invention is added are cultured for 5 days, and then the cells labeled with DPPP are 2, 2 ′. After oxidation with -azobis (2-methylpropionamidine) dihydrochloide, the fluorescence intensity of DPPP in each cell was measured. As a result, compared to the case where 3,5-dihydroxy-4- methoxybenzyl alcohol was not added to the cells, the added cells had a lower fluorescence intensity depending on the concentration. In addition, inhibition of oxidation by the antioxidant substance 3,5-dihydroxy-4-methoxybenzyl alcohol was observed (see FIG. 35).
　以上により、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)は、抗酸化性能を有することが明らかとされた。 From the above, it has been clarified that 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) has antioxidant performance.
　そして、当該3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)は、本発明によって効率よくカキ肉抽出物より抽出できるものであり、もって該カキ肉抽出物により抗酸化剤組成物や抗酸化剤が効率よく多量に回収、製造できることとなる。 The 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) can be efficiently extracted from the oyster meat extract according to the present invention, and thus the oyster meat extract As a result, an antioxidant composition and an antioxidant can be efficiently recovered and produced in large quantities.
　なお、本発明の発明者らは、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4- methoxybenzyl alcohol)の構造を確認するために、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4- methoxybenzyl alcohol)の化学合成を行い、これに成功した。 In order to confirm the structure of 3,5-dihydroxy-4- methoxybenzyl alcohol, the inventors of the present invention used 3,5-dihydroxy-4-methoxybenzyl alcohol. We succeeded in chemical synthesis of benzyl alcohol (3,5-dihydroxy-4- methoxybenzyl alcohol).
　該合成の全工程を図３８に示す。 The whole process of the synthesis is shown in FIG.
　合成された物質につき核磁気共鳴スペクトル（NMR：AMX-500、Bruker、Karlsruhe）、マススペクトル（LXQ、ThermoScientific、Waltham）を測定して、その構造確認を行うものである。 The structure is confirmed by measuring the nuclear magnetic resonance spectrum (NMR: AMX-500, Bruker, Karlsruhe) and mass spectrum (LXQ, Thermo Scientific, Waltham) for the synthesized substance.
　まず、没食子酸メチル（5.00g、27.2mmol）のジメチルホルムアミド（DMF）溶液（45mL）に炭酸カリウム（4.50g、32.6mmol）を加え85℃で１時間撹拌した。その後、氷浴中でヨウ化メチル（4.00g、28.2mmol）3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
を徐々に滴下し30分間撹拌し、さらに室温で24時間撹拌した。反応液をろ過して精製水を加え、酢酸エチルで抽出を行い、分離した酢酸エチル層を飽和食塩水で洗浄後、硫酸ナトリウムで乾燥させた。 First, potassium carbonate (4.50 g, 32.6 mmol) was added to a dimethylformamide (DMF) solution (45 mL) of methyl gallate (5.00 g, 27.2 mmol), and the mixture was stirred at 85 ° C. for 1 hour. Then, methyl iodide (4.00g, 28.2mmol) 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient in an ice bath,
Was gradually added dropwise and stirred for 30 minutes, and further stirred at room temperature for 24 hours. The reaction solution was filtered, purified water was added, extraction was performed with ethyl acetate, and the separated ethyl acetate layer was washed with saturated brine and dried over sodium sulfate.
　抽出液は、濃縮後にシリカゲル・カラムクロマトグラフィー（溶媒：クロロホルム→クロロホルム-酢酸エチル（3:1、v/v））で精製し、4位メトキシ体を2.79g（収率51.9%）得た。 The extract was concentrated and then purified by silica gel column chromatography (solvent: chloroform → chloroform-ethyl acetate (3: 1, v / v)) to obtain 2.79 g (yield 51.9%) of the 4-position methoxy compound.
　条件
1H-NMR(500MHz、CD3OD)δH:7.01(2H、s、H-2、6)、3.85(3H、s、-OMe)、3.82(3H、s、-OMe)；13C-NMR(125MHz、CD3OD)δC:168.5(-C=O)、151.7(C-3、5)、141.2(C-1)、126.5(C-4)、110.1(C-2、6)、60.7(-OMe)、52.5(-OMe)；ESI-ITMS、m/z199[M+H]+、197[M-H]-. conditions
1 H-NMR (500 MHz, CD 3 OD) δ H : 7.01 (2H, s, H-2, 6), 3.85 (3H, s, -OMe), 3.82 (3H, s, -OMe); 13 C- NMR (125 MHz, CD 3 OD) δ C : 168.5 (-C = O), 151.7 (C-3, 5), 141.2 (C-1), 126.5 (C-4), 110.1 (C-2, 6) , 60.7 (-OMe), 52.5 (-OMe); ESI-ITMS, m / z 199 [M + H] + , 197 [MH] − .
　次いで、氷浴中（0℃）で、水素化リチウムアルミニウム（469mg、12.4mmol）のテトラヒドロフラン（THF）溶液（6mL）に、没食子酸メチルの４位メトキシ体（560mg、2.8mmol）のテトラヒドロフラン溶液（4mL）を注意深く滴下した。　その後、混合液を60～65℃で6時間撹拌し、酢酸エチルと10%硫酸水溶液を加えて反応を停止した。反応液に精製水を加え、酢酸エチルで抽出を行い、分離した酢酸エチル層を飽和食塩水で洗浄後に硫酸ナトリウムで乾燥させた。抽出液は、濃縮後に、シリカゲル・カラムクロマトグラフィー（溶媒：クロロホルム-メタノール（50：1、v/v）→クロロホルム-メタノール(50：3、v/v)）で精製し、還元体、すなわち合成3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4- methoxybenzyl alcohol)を175.9mg（収率36.6%）得たのである。 Next, in an ice bath (0 ° C.), a solution of lithium aluminum hydride (469 mg, 12.4 mmol) in tetrahydrofuran (THF) (6 mL) was added to a solution of methyl gallate 4-position methoxy (560 mg, 2.8 mmol) in tetrahydrofuran ( 4 mL) was carefully added dropwise. Thereafter, the mixture was stirred at 60 to 65 ° C. for 6 hours, and the reaction was stopped by adding ethyl acetate and 10% aqueous sulfuric acid. Purified water was added to the reaction solution, extraction was performed with ethyl acetate, and the separated ethyl acetate layer was washed with saturated brine and then dried over sodium sulfate. The extract is concentrated and purified by silica gel column chromatography (solvent: chloroform-methanol (50: 1, v / v) → chloroform-methanol (50: 3, v / v)). As a result, 175.9 mg (yield: 36.6%) of 3,5-dihydroxy-4-methoxybenzyl alcohol was obtained.
　条件
1H-NMR(500MHz、Acetone-d6)δ・H:7.82(2H、br.s、aromatic-OH)、6.40(2H、s、H-2、6)、4.42(2H、s、H-1’)、3.94(1H、br.s、-OH)、3.79(3H、s、-OMe)；13C-NMR(125MHz、Acetone-d6)δC:151.1(C-3、5)、139.4(C-1)、13、5.1(C-4)、106.5(C-2、6)、64.5(C-1’)、60.6(-OMe)；ESI-ITMS、m/z171[M+H]+、153[M-OH]+. conditions
1 H-NMR (500MHz, Acetone -d 6) δ · H: 7.82 (2H, br.s, aromatic-OH), 6.40 (2H, s, H-2,6), 4.42 (2H, s, H- 1 ′), 3.94 (1H, br.s, —OH), 3.79 (3H, s, —OMe); 13 C-NMR (125 MHz, Acetone-d 6 ) δ C : 151.1 (C-3, 5), 139.4 (C-1), 13, 5.1 (C-4), 106.5 (C-2, 6), 64.5 (C-1 '), 60.6 (-OMe); ESI-ITMS, m / z171 [M + H ] + , 153 [M-OH] + .
　（3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)とその合成品の物性パラメータの比較）
3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)とその合成品との間では、上記の1H-NMR、13C-NMR、ESI-MSの各種スペクトルデータが一致し、HPLCの保持時間及び薄層クロマトグラフィーの移動度が一致したことにより、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)であることが確認できる。 (Comparison of physical parameters of 3,5-dihydroxy-4-methoxybenzyl alcohol and its synthetic product)
Between 1, 5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) and its synthetic products, various spectra of the above 1 H-NMR, 13 C-NMR, and ESI-MS The data was consistent and the retention time of HPLC and mobility of thin layer chromatography were consistent, indicating that it was 3,5-dihydroxy-4-methoxybenzyl alcohol. I can confirm.
　前述のごとく、カキより得られた抗酸化物質、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4- methoxybenzyl alcohol)、は、高度に水酸化された芳香族化合物といいうる。 As mentioned above, 3,5-dihydroxy-4- methoxybenzyl alcohol, an antioxidant obtained from oysters, is a highly hydroxylated aromatic compound. sell.
　これまでにも、水酸化された芳香族、即ちフェノール化合物においては、コーヒー酸に代表されるようなクロロゲン酸、ニグニン類やフラボノイドなど、多くの物質が抗酸化活性を有することが報告されている。 So far, in hydroxylated aromatic, ie, phenolic compounds, it has been reported that many substances such as chlorogenic acid typified by caffeic acid, ignnins and flavonoids have antioxidant activity. .
　これらの物質は、ペルオキシド、特にペルオキシラジカル（ROO・）の捕捉剤として働くことにより抗酸化活性を発揮することが知られている。しかして、本発明で同定された抗酸化物質は、ラジカル消去能を観察するORACやAAPHを用いる細胞実験で高い抗酸化活性を示している。 These substances are known to exhibit antioxidant activity by acting as a scavenger for peroxides, particularly peroxy radicals (ROO.). Thus, the antioxidant substance identified in the present invention exhibits high antioxidant activity in cell experiments using ORAC or AAPH for observing radical scavenging ability.
　このことは、当該物質がラジカル捕捉剤として抗酸化能を発揮する可能性を強く示唆したものともいいうる。 This can be said to strongly suggest the possibility that the substance exhibits antioxidant ability as a radical scavenger.
　本発明によって精製された当該物質は、両親媒性であるが、数少ない水溶性抗酸化剤の一つとして広く用いられるL(+)-アスコルビン酸と比較してORAC値が３倍大きかったことは、当該物質の抗酸化剤としての効果を強く期待させるものである。当該物質が用量依存的にLDLの酸化を抑制したことは、同物質がLDLの酸化防止を通じて抗動脈硬化作用を発揮する効果を示唆している。 The substance purified according to the present invention is amphiphilic, but the ORAC value was three times higher than that of L (+)-ascorbic acid, which is widely used as one of the few water-soluble antioxidants. The effect of the substance as an antioxidant is highly expected. The fact that the substance suppressed LDL oxidation in a dose-dependent manner suggests that the substance exerts an anti-arteriosclerotic effect through the prevention of LDL oxidation.
　一方、生細胞の酸化をリアルタイムに観察する目的で、cis-parinaricacid(PnA)、fluoresceinatedphosphoethanolamine、undecylamine-fluoresceinなどのプローブが開発されてきた。中でもPnAが生細胞の酸化を観察するプローブとしてよく使われているが、PnAはしばしば細胞毒性があり、細胞の生理学的活性に影響を及ぼすことが報告されている。 On the other hand, probes such as cis-parinaricacid (PnA), fluoresceinatedphosphoethanolamine, and undecylamine-fluorescein have been developed for the purpose of observing live cell oxidation in real time. Among them, PnA is often used as a probe for observing the oxidation of living cells, but PnA is often cytotoxic and has been reported to affect the physiological activity of cells.
　本発明で使用したDPPPは、細胞増殖や細胞毒性などに少なくとも3日間は影響を与えず、DPPP及び酸化されたDPPPは生細胞の細胞膜に局在し、少なくても２日間は安定であることが報告されている。このDPPP自体は蛍光を発しないが、酸化されたDPPPは蛍光を発する。このDPPPを用い、従来、生細胞の過酸化脂質を観察する方法が確立し、ヒト単球系の浮遊細胞(U937)を用いて、ビタミンＥの抗酸化力を確認している。 DPPP used in the present invention does not affect cell proliferation or cytotoxicity for at least 3 days, and DPPP and oxidized DPPP are localized in the cell membrane of living cells and stable for at least 2 days. Has been reported. The DPPP itself does not fluoresce, but the oxidized DPPP fluoresces. Conventionally, a method for observing lipid peroxides in living cells using this DPPP has been established, and the antioxidant activity of vitamin E has been confirmed using human monocyte-based floating cells (U937).
　本発明において、当該抗酸化物質が肝臓由来の株化細胞であるC3A細胞の酸化を有意に抑制したことは、本発明による当該物質が少なくとも肝細胞内においても抗酸化活性を発揮できることを示唆するのである。この結果は、上述のLDL酸化抑制と併せて、本発明物質の抗酸化剤としての効果を大きく期待させる。 In the present invention, the fact that the antioxidant substance significantly suppressed the oxidation of C3A cells, which are liver-derived cell lines, suggests that the substance according to the present invention can exert an antioxidant activity even at least in hepatocytes. It is. This result, together with the above-described inhibition of LDL oxidation, greatly increases the effect of the substance of the present invention as an antioxidant.
　酸化ストレスが関連する疾患として、従来は動脈硬化性疾患に大きな関心が向けられてきたが、近年では非アルコール性脂肪性肝炎(NASH)などの異所性脂肪蓄積症にも注目が集まりつつある。NASHでは、活性酸素が肝細胞壊死や炎症性サイトカイン生産、肝線維化に関係することが知られている。またNASHでは血中の酸化LDL濃度が高いことが報告されている。さらに、NASHの炎症性サイトカイン産生亢進やコラーゲン産生亢進に関与する肝星細胞は、酸化LDLにより活性化されることが報告されている。 As a disease related to oxidative stress, attention has been focused on arteriosclerotic diseases, but in recent years, ectopic fat accumulation diseases such as non-alcoholic steatohepatitis (NASH) are also attracting attention. . In NASH, it is known that active oxygen is related to hepatocyte necrosis, inflammatory cytokine production, and liver fibrosis. NASH has also been reported to have high levels of oxidized LDL in the blood. Furthermore, it has been reported that hepatic stellate cells involved in the enhancement of inflammatory cytokine production and collagen production of NASH are activated by oxidized LDL.
　本発明で見出されたカキの新規抗酸化物質がNASHの予防に貢献できるものと大きく期待できる。 It can be greatly expected that the new antioxidant of oysters found in the present invention can contribute to the prevention of NASH.
　このように、本発明では、カキより新規抗酸化物質を見出し、その化学構造を3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)と決定した。さらには、その化学合成法をも確定した。 Thus, in the present invention, a novel antioxidant was found from oysters, and its chemical structure was determined to be 3,5-dihydroxy-4-methoxybenzyl alcohol. Furthermore, the chemical synthesis method was also confirmed.
　さらに、本発明による当該物質のORAC値は、その精製物が1.24±0.3、5μmolTE/μmol、その合成物が1.47±0.40μmolTE/μmolであり、水溶性抗酸化物質のクロロゲン酸とL(+)-アスコルビン酸の中間の抗酸化能の強さであった。 Further, the ORAC value of the substance according to the present invention is 1.24 ± 0.3, 5 μmol TE / μmol for the purified product, 1.47 ± 0.40 μmol TE / μmol for the synthesized product, and chlorogenic acid and L (+) of the water-soluble antioxidant substance. -It was an intermediate antioxidative strength of ascorbic acid.
　また、ヒトLDLの金属酸化に対して、本発明による当該物質3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4- methoxybenzyl alcohol)は用量依存的に抗酸化能を示し、C3A細胞を用いた抗酸化能実験においても、当該物質は用量依存的に抗酸化能を示したことを付言する。 In addition, against the metal oxidation of human LDL, the substance 3, 5-dihydroxy-4-methoxybenzyl alcohol (3, 5-dihydroxy-4- methoxybenzyl alcohol) according to the present invention shows an antioxidant ability in a dose-dependent manner, It is added that the substance showed an antioxidant ability in a dose-dependent manner in the antioxidant ability experiment using C3A cells.
　次に、本件発明者らは、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を高濃度含有するマガキの抽出物をNASHモデルマウスに投与し、肝臓の保護作用を有するか否かの実験を行った。 Next, the present inventors administered a oyster extract containing a high concentration of 3,5-dihydroxy-4-methoxybenzylmethoxyalcohol to a NASH model mouse, and the liver An experiment was conducted to determine whether or not it has a protective action.
　しかして、図１乃至図２２に本発明を説明する実験データなどを示す。 However, FIG. 1 to FIG. 22 show experimental data for explaining the present invention.
　図１から理解されるように、マウスのNC群には普通食（MF）の餌料を与え、マウスのHF+群には高脂肪食（HFD-60）と3、5-ジヒドロキシ-4-メトキシベンジルアルコールを高濃度含有するマガキの抽出物が5%含有された餌料を与え、マウスのHF-群には高脂肪食（HFD-60）のみの餌料を与えた。
なお、餌料はマウスが自由に食べる分だけ与えるものとした。 As can be seen from FIG. 1, the NC group of mice is fed a normal diet (MF), and the HF + group of mice is fed with a high fat diet (HFD-60) and 3,5-dihydroxy-4-methoxybenzyl. A diet containing 5% oyster extract containing a high concentration of alcohol was given, and the HF-group of mice was fed only a high fat diet (HFD-60).
It should be noted that the food is given only for the amount that the mouse eats freely.
　生後４週目のマウスを実験開始時とし、実験開始２０週目から２日おきに尾静脈に0.2mgの酸化LDLの注射を計８回注射する。最終の酸化LDLの注射後、１２時間後に屠殺する。屠殺の１６時間前に餌止めを行い、ジエチルエーテルにより麻酔し、脱血後、サンプルを採取する。 4 weeks old mice are set at the start of the experiment, and 0.2 mg of oxidized LDL is injected into the tail vein every 2 days from the 20th week of the experiment. Sacrifice 12 hours after the final injection of oxidized LDL. Food is stopped 16 hours before sacrifice, anesthetized with diethyl ether, and after blood removal, a sample is taken.
　次に、図２に分析項目を示す。 Next, Fig. 2 shows the analysis items.
　分析項目として、餌料は摂餌量(g)を、外観はマウスの体重(g)、肝重量(g)及び体重当たりの肝重量(%)を、肝臓の組織学的観察は脂肪蓄積度、バルーニング度、炎症度及び線維化度を、肝臓は中性脂肪(mg/dl)、総コレステロール(mg/dl)、遊離脂肪酸(mEq/L)及びTBARS（μM）を、血漿はAST（U/L）、ALT（U/L）、グルコース(mg/dL)、インスリン(μg/L)、インスリン抵抗性指数（HOMA-IR）、中性脂肪(mg/dL)、総コレステロール(mg/dL)及び遊離脂肪酸(mEq/L)を挙げて分析する。 As analysis items, food consumption (g), appearance is mouse body weight (g), liver weight (g) and liver weight per body weight (%), liver histological observation is fat accumulation, Ballooning degree, inflammation degree and fibrosis degree, liver shows neutral fat (mg / dl), total cholesterol (mg / dl), free fatty acid (mEq / L) and TBARS (μM), plasma shows AST (U / L), ALT (U / L), glucose (mg / dL), insulin (μg / L), insulin resistance index (HOMA-IR), neutral fat (mg / dL), total cholesterol (mg / dL) And free fatty acid (mEq / L).
　図３に各マウスのHF+群とHF-群の摂餌量(g)を示す。 Fig. 3 shows the food intake (g) of the HF + group and HF- group of each mouse.
　摂餌量は、最初に与えた餌量と３、４日後の餌量を計測して、最初に与えた餌量から３、４日後の餌量を引いた量が摂餌量となる。 The amount of food intake is the amount obtained by measuring the amount of food initially given and the amount of food after 3 and 4 days, and subtracting the amount of food given 3 and 4 days from the amount of food initially given.
　図３の（ａ）は実験開始１～２３週間の摂餌量を表し、図３の（ｂ）は各マウスのHF+群とHF-群における生涯の摂餌量を表している。ここで、図３の（ａ）は実験開始１～１０週目ごろまではHF-群、HF+群の各マウスにおいて、標準時偏差が大きく出ているが、これはマウスがエサを容器の外に出し、まき散らすためである。しかしながら、図３の（ｂ）より各マウスのHF+群とHF-群における生涯の摂餌量は統計的に有意ではなく、生涯の摂餌量は同じと考えられる。 (A) in FIG. 3 represents the food intake for 1 to 23 weeks from the start of the experiment, and (b) in FIG. 3 represents the lifetime food intake of each mouse in the HF + group and HF- group. Here, (a) in FIG. 3 shows that the standard time deviation is large in each mouse of the HF− group and the HF + group from the first to the tenth week from the start of the experiment. This is because the mouse puts the food outside the container. It is for putting out and spreading. However, from FIG. 3 (b), the lifetime food intake of each mouse in the HF + group and the HF− group is not statistically significant, and the lifetime food intake is considered to be the same.
　図４にマウスの外観と肝臓の色調を分析した結果を示す。 Fig. 4 shows the results of analyzing the appearance of the mouse and the color of the liver.
　マウスのNC群とマウスのHF+群又はマウスのHF-群の外観と肝臓の色調を比較してみると、HF+群とHF-群の生涯の摂餌量は同じであるにもかかわらず、HF+群の外観と肝臓の色調はNC群に近いことが確認された。すなわち、マウスのHF+群に抗肥満作用が確認できた。 Comparing the appearance and liver color of the NC group of mice and the HF + group of mice or the HF- group of mice, HF + The appearance of the group and the color of the liver were confirmed to be close to those of the NC group. That is, an anti-obesity action was confirmed in the HF + group of mice.
　図５に実験開始１～２３週間のNC群、HF-群及びHF+群の各マウスにおける体重の変化を分析した結果を示す。 FIG. 5 shows the results of analysis of changes in body weight of mice in the NC group, HF− group and HF + group for 1 to 23 weeks from the start of the experiment.
　摂餌後６週目よりHF-群のマウスの体重とHF+群のマウスの体重では、HF+群のマウスの体重の方が軽くなっていることが確認された。すなわち、摂餌後６週目よりマウスのHF+群に抗肥満作用が確認できた。 From the 6th week after feeding, it was confirmed that the weight of the mice in the HF + group was lighter than that of the mice in the HF + group and the mice in the HF + group. That is, the anti-obesity action was confirmed in the HF + group of mice from 6 weeks after feeding.
　NC群、HF-群及びHF+群の各マウスにおける、図６の（ａ）はマウスの体重、図６の（ｂ）は肝重量、図６の（ｃ）は体重当たりの肝重量を分析した結果を示す。 In each mouse of the NC group, the HF− group, and the HF + group, FIG. 6 (a) is the body weight of the mouse, FIG. 6 (b) is the liver weight, and FIG. 6 (c) is the liver weight per body weight. Results are shown.
　図６の（ｃ）よりHF+群の体重当たりの肝重量がHF-群の体重当たりの肝重量より減少していることが確認された。すなわち、マウスのHF+群に抗肥満作用が確認できたのである。 6 (c), it was confirmed that the liver weight per body weight of the HF + group was smaller than the liver weight per body weight of the HF− group. That is, an anti-obesity action was confirmed in the HF + group of mice.
　図７は病理学的観察におけるスコアの基準について、図８は各判定の正常細胞及び病的細胞の写真を示す。 FIG. 7 shows score criteria in pathological observation, and FIG. 8 shows photographs of normal cells and pathological cells in each determination.
　図７の表中のスコア0は図８に示す正常細胞に該当し、スコア２－３は図８に示す病的細胞に該当する。スコアの判断基準は「脂肪蓄積度」、「バーニング度」、「炎症度」及び「線維化度」で判定する。 In the table of FIG. 7, score 0 corresponds to the normal cell shown in FIG. 8, and score 2-3 corresponds to the pathological cell shown in FIG. The criteria for determining the score are “Fat accumulation degree”, “Burning degree”, “Inflammation degree”, and “Fibrosis degree”.
　「脂肪蓄積度」においては脂肪染色で赤く染まる占有率を画像解析ソフトによって求め、それぞれの占有率で判定する。 In “Fat accumulation degree”, the occupancy ratio that is dyed red by fat staining is obtained by image analysis software, and is determined by each occupancy ratio.
　「バーニング度」はHE染色で膨れている細胞が１視野中にどのくらいの頻度か(点在か、汎細葉性か)を数え判定する。 “Burning degree” is determined by counting how often cells swelled by HE staining are scattered in one visual field (whether they are scattered or panlobular).
　「炎症度」はHE染色で炎症があると青色に染まるリンパ球やマクロファージの浸潤が肝臓にみられるので、１視野中にどのくらいの病巣があるかを数え判定する。 “Inflammation” is determined by counting how many lesions are present in one field of view because infiltration of blue lymphocytes and macrophages is observed in the liver when inflammation is detected by HE staining.
　「線維化度」は線維を青く染めるマッソントリクローム染色で細胞周辺と静脈だけなのか、又はそれらが繋がっているかの病態で判定する。 The “fibrosis degree” is determined by Masson trichrome staining that stains the fibers blue, based on the pathological condition of whether the cell periphery and veins are connected, or whether they are connected.
　図９にNC群、HF-群及びHF+群の各マウスの肝臓の病理学的観察の結果を示す。 FIG. 9 shows the results of pathological observation of the livers of mice in the NC group, the HF− group, and the HF + group.
　各判定結果より、HF+群の肝臓において脂肪蓄積度、バーニング度、炎症度及び線維化度の減少が確認できた。 From each determination result, it was confirmed that the fat accumulation, burning, inflammation, and fibrosis were decreased in the liver of the HF + group.
　図１０にNC群、HF-群及びHF+群の各マウスの肝臓の各脂質の濃度を分析した結果を示す。 FIG. 10 shows the result of analyzing the concentration of each lipid in the liver of each mouse of NC group, HF-group and HF + group.
　各判定結果より、HF+群の肝臓において中性脂肪、総コレステロール、遊離脂肪酸及び脂質の過酸化度(TBARS)の減少が確認できた。 From the results of each determination, it was confirmed that neutral fat, total cholesterol, free fatty acids and lipid peroxidation (TBARS) decreased in the liver of the HF + group.
　図１１にNC群、HF-群及びHF+群の各マウス血漿中のAST活性、ALT活性について分析した結果を示す。 FIG. 11 shows the results of analysis of AST activity and ALT activity in mouse plasma of NC group, HF− group and HF + group.
　AST活性、ALT活性の判定結果より、HF+群の血漿においてAST、ALT活性の減少が確認できた。 From the determination results of AST activity and ALT activity, a decrease in AST and ALT activity was confirmed in the plasma of the HF + group.
　図１２にNC群、HF-群及びHF+群の各マウス血漿中のグルコース、インスリン濃度及びインスリン抵抗性指数について分析した結果を示す。 FIG. 12 shows the results of analysis of glucose, insulin concentration, and insulin resistance index in the plasma of each mouse in the NC group, HF− group, and HF + group.
　ここで、インスリン抵抗性指数は、
インスリン抵抗性指数＝空腹時インスリン値（ｍU/ml）×空腹時血糖値(mmol/L)／22.5
　の数式が使用される。 Where the insulin resistance index is
Insulin resistance index = fasting insulin level (mU / ml) x fasting blood glucose level (mmol / L) /22.5
The following formula is used.
　各判定結果より、HF+群の血漿においてグルコース、インスリン濃度及びインスリン抵抗性指数の減少が確認できた。 From each determination result, it was confirmed that glucose, insulin concentration and insulin resistance index decreased in plasma of HF + group.
　図１３にNC群、HF-群及びHF+群の各マウスの血漿の各脂質の濃度を分析した結果を示す。 FIG. 13 shows the result of analyzing the concentration of each lipid in the plasma of each mouse in the NC group, HF-group and HF + group.
　各判定結果より、HF+群の血漿において中性脂肪、総コレステロール及び遊離脂肪酸の減少が確認できた。 From the results of each determination, it was confirmed that neutral fat, total cholesterol and free fatty acids were reduced in the plasma of the HF + group.
　図１４、図１５に肝臓における炎症反応に関する遺伝子について示す。 FIG. 14 and FIG. 15 show genes related to the inflammatory reaction in the liver.
　図１５を参照して説明すると、NASHモデルマウスの肝臓において、活性酸素種（ROS）により肝細胞が炎症を受けると、そこにマクロファージやリンパ球が集合してくるので、マクロファージの遊走を促進するサイトカインのIL-6やTNF-αの遺伝子が上昇するのである。またマクロファージやリンパ球を特異的に認識するマーカーであるF4/80やCD3の遺伝子が上昇する。 Referring to FIG. 15, when hepatocytes are inflamed by reactive oxygen species (ROS) in the liver of a NASH model mouse, macrophages and lymphocytes gather there, thereby promoting macrophage migration. The cytokine IL-6 and TNF-α genes are elevated. In addition, F4 / 80 and CD3 genes, which are markers that specifically recognize macrophages and lymphocytes, are elevated.
　図１４はNC群、HF-群及びHF+群の各マウスの炎症反応に関する肝臓の遺伝子を、図１４（ａ）はTNF-αの遺伝子について、図１４（ｂ）はIL-6の遺伝子について、図１４（ｃ）はマクロファージのマーカーであるF4/80の遺伝子について、図１４（ｄ）はリンパ球のマーカーであるCD3の遺伝子について分析した結果である。 FIG. 14 shows liver genes related to the inflammatory reaction of each mouse in the NC group, HF− group, and HF + group, FIG. 14A shows the TNF-α gene, and FIG. 14B shows the IL-6 gene. FIG. 14 (c) shows the result of analysis of the F4 / 80 gene, which is a macrophage marker, and FIG. 14 (d) shows the result of analysis of the CD3 gene, which is a lymphocyte marker.
　各判定結果より、HF+群はHF-群と比べて、これらの遺伝子の発現が低下していた。すなわち、HF+群の肝臓において炎症反応に関連する遺伝子の減少が確認できた。 From each determination result, the expression of these genes was decreased in the HF + group compared to the HF- group. That is, a decrease in genes related to the inflammatory reaction was confirmed in the liver of the HF + group.
　図１６、図１７に肝臓におけるアポトーシスに関する遺伝子について示す。 16 and 17 show genes related to apoptosis in the liver.
　図１７を参照して説明すると、NASHモデルマウスの肝臓において、活性酸素種（ROS）により肝細胞が炎症を受けると、アポトーシスによる細胞死が観察される。これに伴い、肝臓において、アポトーシスを促進する遺伝子であるBaxの上昇し、アポトーシスを抑制する遺伝子であるBcl2, Bcl-xl, p53 の減少が観察される。 Referring to FIG. 17, when hepatocytes are inflamed by reactive oxygen species (ROS) in the liver of a NASH model mouse, cell death due to apoptosis is observed. Along with this, in the liver, Bax, which is a gene that promotes apoptosis, is increased, and decreases in Bcl2, Bcl-xl, and p53 抑制 す る, which are genes that suppress apoptosis, are observed.
　図１６はNC群、HF-群及びHF+群の各マウスのアポトーシスに関する肝臓の遺伝子を、図１６（ａ）はBax遺伝子について、図１６（ｂ）はBcl-xl遺伝子について、図１６（ｃ）はBcl2遺伝子について、図１６（ｄ）はp53遺伝子について分析した結果である。 FIG. 16 shows liver genes related to apoptosis in mice of NC group, HF− group and HF + group, FIG. 16A shows Bax gene, FIG. 16B shows Bcl-xl gene, and FIG. Fig. 16 (d) shows the results of analysis for the Bcl2 gene and Fig. 16 (d) for the p53 gene.
　各判定結果より、HF+群はHF-群と比べて、Baxの低下やBcl2, Bcl-xl, p53の上昇がみられた。すなわち、HF+群の肝臓においてアポトーシスに関連する遺伝子の減少及び抗アポトーシスに関連する遺伝子の増加が確認できた。 From the judgment results, the HF + group showed a decrease in Bax and an increase in Bcl2, Bcl-xl, and p53 compared to the HF-group. That is, in the liver of the HF + group, a decrease in genes related to apoptosis and an increase in genes related to anti-apoptosis were confirmed.
　図１８、図１９に肝臓におけるアポトーシスに関する遺伝子について示す。 18 and 19 show genes related to apoptosis in the liver.
　図１９を参照して説明すると、NASHモデルマウスの肝臓において、活性酸素種（ROS）により肝細胞が炎症を受けると、線維化が観察される。これに伴い、肝臓において、線維化を促進する遺伝子であるCOL1a2，COL3，COL4，TGFβ1，TGFβ2，TIMP1，αSMAの上昇が観察される。 Referring to FIG. 19, fibrosis is observed when hepatocytes are inflamed by reactive oxygen species (ROS) in the liver of a NASH model mouse. Along with this, an increase in COL1a2, COL3, COL4, TGFβ1, TGFβ2, TIMP1, and αSMA, which are genes that promote fibrosis, is observed in the liver.
　図１８はNC群、HF-群及びHF+群の各マウスの線維化に関する肝臓の遺伝子を、図１８（ａ）はCOL1a2の遺伝子について、図１８（ｂ）はCOL3の遺伝子について、図１８（ｃ）はCOL4の遺伝子について、図１８（ｄ）はTGFβ1の遺伝子について、図１８（ｅ）はTGFβ2の遺伝子について、図１８（ｆ）はTIMP1の遺伝子について、図１８（ｇ）はαSMAの遺伝子について分析した結果である。 FIG. 18 shows liver genes related to fibrosis of NC group, HF− group, and HF + group mice, FIG. 18A shows COL1a2 genes, FIG. 18B shows COL3 genes, and FIG. ) For the COL4 gene, FIG. 18 (d) for the TGFβ1 gene, FIG. 18 (e) for the TGFβ2 gene, FIG. 18 (f) for the TIMP1 gene, and FIG. 18 (g) for the αSMA gene. It is the result of analysis.
　各判定結果より、HF+群はHF-群と比べて、COL1a2，COL3，COL4，TGFβ1，TGFβ2，TIMP1，αSMAの低下がみられた。すなわち、HF+群の肝臓において線維化に関連する遺伝子の減少が確認できた。 From the judgment results, the HF + group showed lower COL1a2, COL3, COL4, TGFβ1, TGFβ2, TIMP1, and αSMA than the HF- group. That is, a decrease in genes related to fibrosis was confirmed in the liver of the HF + group.
　図２０に分析項目の結果のまとめを示す。 Fig. 20 shows a summary of the analysis item results.
　図２１、図２２に示すように、3、5-ジヒドロキシ-4-メトキシベンジルアルコールを高濃度含有するマガキからの抽出物は、NASHモデルマウスにおいて、抗肥満、抗脂肪肝、抗炎症、抗線維化、抗アポトーシス、抗酸化、血糖値低下、血漿脂質低下などの優れた効果が確認できたのである。 As shown in FIGS. 21 and 22, an extract from a oyster containing a high concentration of 3,5-dihydroxy-4-methoxybenzyl alcohol was used in anti-obesity, anti-fatty liver, anti-inflammation, anti-fibrosis in a NASH model mouse. Excellent effects such as oxidization, anti-apoptosis, anti-oxidation, blood glucose level lowering, and plasma lipid lowering were confirmed.
　さらに、実験データの説明図（図３９乃至図５０）を示して説明する。 Further explanation will be given by showing explanatory diagrams (FIGS. 39 to 50) of the experimental data.
　肝臓の炎症反応の特徴であるリンパ球とマクロファージの浸潤を抗-CD3抗体(リンパ球に特異的な抗体)と抗-F4/80 (マクロファージに特異的な抗体)を用いて免疫組織化学的観察を行った。 Immunohistochemical observation of infiltration of lymphocytes and macrophages, characteristic of liver inflammatory response, using anti-CD3 antibody (antibody specific for lymphocytes) and anti-F4 / 80- (antibody specific for macrophages) Went.
　図３９には両抗体を用いたNC、HF+、HF-群の代表的な染色像を示す。HF+においては、両抗体により点在している染色像が見られた。HF-群ではNC群と比較し、リンパ球とマクロファージの肝臓への浸潤が観察された。一方、HF+群ではHF-群と比較し、両血球とも浸潤はほとんど認められなかった。 FIG. 39 shows representative stained images of NC, HF + and HF− groups using both antibodies. In HF +, stained images interspersed with both antibodies were observed. In the HF-group, infiltration of lymphocytes and macrophages into the liver was observed compared to the NC group. On the other hand, in the HF + group, almost no infiltration was observed in both blood cells compared to the HF- group.
　図４０では図３９で行った両免疫染色像の占有率をImageJを用いて定量化したグラフを示す。HF-群ではNC群と比較し、統計学的に優位に染色像の増加が観察されたが、HF+群ではHF-群と比較し、統計学的に優位な減少が観察された。これらの結果より、前述のマガキの抽出物によるNASHモデルマウスの抗炎症作用が観察された。 FIG. 40 shows a graph in which the occupation ratio of both immunostained images performed in FIG. 39 is quantified using ImageJ. The HF- group showed a statistically significant increase in staining images compared to the NC group, but the HF + group showed a statistically significant decrease compared to the HF- group. From these results, the anti-inflammatory action of the NASH model mouse by the aforementioned oyster extract was observed.
　肝臓のアポトーシスの特徴であるDNAの断片化をTUNEL assayを用いて観察を行った。図４１ではTUNEL assayにおける代表的な染色像と染色数のグラフを示す。HF-群においては、細胞の核が染色している像が見られた。染色細胞数のグラフから、HF-群ではNC群と比較し、統計学的に優位にDNAの断片化を起こしている細胞数の増加が観察されたが、HF+群ではHF-群と比較し、統計学的に優位な減少が観察された。これらの結果より、前述のマガキの抽出物によりNASHモデルマウスの抗アポトーシス作用が観察された。 The DNA fragmentation characteristic of liver apoptosis was observed using TUNEL assay. FIG. 41 shows a representative staining image and the number of staining in TUNEL assay. In the HF-group, the cell nucleus was stained. From the graph of the number of stained cells, the HF- group showed a statistically significant increase in the number of cells causing DNA fragmentation compared to the NC group, but the HF + group compared to the HF- group. A statistically significant decrease was observed. From these results, the anti-apoptotic action of NASH model mice was observed with the above-mentioned oyster extract.
　肝臓の線維化の特徴であるコラーゲンの蓄積を抗-コラーゲン抗体を用いて観察を行った。図４２では抗-コラーゲン抗体を用いたNC、HF+、HF+群の代表的な免疫組織化学染色像、及びImageJを用いて定量化したグラフを示す。HF-群においては、細静脈周囲に顕著なコラーゲンの染色像が見られた。HF-群ではNC群と比較し、統計学的に優位にコラーゲンの蓄積を起こしている領域の増加が観察されたが、HF+群ではHF-群と比較し、統計学的に優位な減少が観察された。これらの結果より、マガキの抽出物によりNASHモデルマウスの抗線維化作用が観察された。 The accumulation of collagen, which is characteristic of liver fibrosis, was observed using an anti-collagen antibody. FIG. 42 shows representative immunohistochemically stained images of NC, HF +, and HF + groups using anti-collagen antibodies, and a graph quantified using ImageJ. In the HF-group, a marked collagen staining image was seen around the venule. In the HF- group, an increase in the area where collagen accumulation was statistically significant was observed compared to the NC group, but in the HF + group, a statistically significant decrease was observed compared to the HF- group. Observed. From these results, the antifibrotic effect of NASH model mice was observed with the oyster extract.
　肝臓における酸化の状態をDNAの酸化マーカーである8OHｄGの抗体を用いて、タンパク質マーカーでるdityrosineの抗体を用いて観察を行った。図４３には両抗体を用いたNC、HF+、HF+群の代表的な免疫組織化学染色像を示す。抗-8OHｄG抗体を用いた染色像では細胞の核に、抗-dityrosine抗体を用いた染色像では細胞質に染色像が見られた。HF-群ではNC群と比較し、両抗体とも染色領域の増加が観察された。一方、HF+群ではHF-群と比較し、両抗体とも染色領域の減少が観察された。 The state of oxidation in the liver was observed using an 8OHdG antibody, which is a DNA oxidation marker, and a dityrosine antibody, which is a protein marker. FIG. 43 shows representative immunohistochemically stained images of NC, HF +, and HF + groups using both antibodies. The stained image using the anti-8OHdG antibody showed a stained image in the cell nucleus, and the stained image using the anti-dityrosine antibody showed a stained image in the cytoplasm. In the HF-group, an increase in the staining area was observed for both antibodies compared to the NC group. On the other hand, in the HF + group, a decrease in the staining area was observed for both antibodies compared to the HF- group.
　図４４では図４３で行った抗-8OHｄG抗体と抗-dityrosine抗体での免疫染色像の占有率をImageJを用いて定量したグラフ、並びに脂質の酸化状態であるTBARS量のグラフを示す。両抗体での免疫染色像の占有率、及びTBARS量とも、HF-群ではNC群と比較し、統計学的に優位な増加が観察された。一方、HF+群ではHF-群と比較し、統計学的に優位な減少が観察された。 44 shows a graph obtained by quantifying the occupation ratio of the immunostained image with the anti-8OHdG antibody and the anti-dityrosine antibody performed in FIG. 43 using ImageJ, and a graph of the amount of TBARS which is the oxidation state of lipid. A statistically significant increase was observed in the HF-group compared to the NC group in both the occupancy ratio of the immunostained images with both antibodies and the amount of TBARS. On the other hand, a statistically significant decrease was observed in the HF + group compared to the HF- group.
　図４５のイラストに示すように、前記マガキの抽出物によりNASHモデルマウスの肝臓におけるDNA、脂質、及びタンパク質における抗酸化性が観察された。 As shown in the illustration of FIG. 45, antioxidant properties of DNA, lipids and proteins in the liver of NASH model mice were observed with the oyster extract.
　図４６のイラストに示すように、脂肪肝やインスリン抵抗性に関わる転写因子PPARγ・と受容体CD36を示す。先行研究により、以下の報告がある。
１．ヒトの脂肪肝におけるPPARγの主な転写産物はCD36であった。
２．NAFLD患者において、インスリン抵抗性や高インスリン血症にCD36が深く関与していた。 As shown in the illustration of FIG. 46, the transcription factor PPARγ · involved in fatty liver and insulin resistance and the receptor CD36 are shown. According to previous research, there are the following reports.
1. The main transcript of PPARγ in human fatty liver was CD36.
2. CD36 was deeply involved in insulin resistance and hyperinsulinemia in NAFLD patients.
　これらのことから、PPARγ、CD36の発現量を観察することは脂肪肝やインスリン抵抗性を観察するうえでも大変重要である。 Therefore, observing the expression levels of PPARγ and CD36 is very important for observing fatty liver and insulin resistance.
　肝臓での転写因子PPARγを抗- PPARγ・抗体、受容体CD36を抗- CD36抗体を用いて観察を行った。図４７には両抗体を用いたNC、HF+、HF+群の代表的な免疫組織化学染色像を示す。抗- PPARγ抗体を用いた染色像では細胞の核に、抗-CD36抗体を用いた染色像では細胞質に染色像が見られた。HF-群の肝臓ではNC群と比較し、両抗体とも染色領域の増加が観察された。一方、HF+群ではHF-群と比較し、両抗体とも染色領域の減少が観察された。 The transcription factor PPARγ in the liver was observed using an anti- PPARγ • antibody, and the receptor CD36 was observed using an anti- CD36 antibody. FIG. 47 shows representative immunohistochemically stained images of NC, HF +, and HF + groups using both antibodies. Stained images using the anti- PPARγ antibody showed a stained image in the cell nucleus, and stained images using the anti-CD36 antibody showed a stained image in the cytoplasm. In the liver of the HF- group, an increase in the staining area was observed for both antibodies compared to the NC group. On the other hand, in the HF + group, a decrease in the staining area was observed for both antibodies compared to the HF- group.
　図４８では図４７で行った免疫染色像の占有率をImageJを用いて定量化したグラフを示す。HF-群ではNC群と比較し、占有率の増加が観察されたが、HF+群ではHF-群と比較し、統計学的に優位な占有率の減少が観察された。これらの結果より、前記したマガキの抽出物によりNASHモデルマウスの肝臓において、統計学的に優位な両タンパク質発現量の減少が観察された。 48 shows a graph in which the occupation ratio of the immunostained image performed in FIG. 47 is quantified using ImageJ. In the HF- group, an increase in the occupation rate was observed compared with the NC group, but in the HF + group, a statistically significant decrease in the occupation rate was observed compared with the HF- group. From these results, a statistically significant decrease in the expression level of both proteins was observed in the liver of NASH model mice by the above-described oyster extract.
　図４９では肝臓での転写因子PPARγ、受容体CD36の遺伝子発現量を示す。HF-群ではNC群と比較し、両遺伝子発現量の増加が観察されたが、HF+群ではHF-群と比較し、統計学的に優位な両遺伝子発現量の減少が観察された。図47-49の結果より、マガキの抽出物によりNASHモデルマウスの肝臓において、統計学的に優位なPPARγ、CD36の発現量の減少が観察された。 FIG. 49 shows gene expression levels of transcription factor PPARγ and receptor CD36 in the liver. In the HF-group, an increase in both gene expression levels was observed compared to the NC group, but in the HF + group, a statistically significant decrease in both gene expression levels was observed compared to the HF-group. From the results of FIGS. 47-49, statistically significant reductions in the expression levels of PPARγ and CD36 were observed in the livers of NASH model mice by the oyster extract.
　図５０に図３９乃至図４９における分析項目の結果のまとめを示す。
  FIG. 50 shows a summary of the analysis item results in FIGS.
１        　抽出容器
２        　抽出用溶液
３        　生カキ肉
４        　エタノール溶液
５        　酢酸エチル
６        　濃縮液
７        　沈殿物
８        　上澄み液
９        　上澄み液の濃縮液
１０      　希釈液
１０ａ　　　水層 DESCRIPTION OF SYMBOLS 1 Extraction container 2 Extraction solution 3 Raw oyster meat 4 Ethanol solution 5 Ethyl acetate 6 Concentrate 7 Precipitate 8 Supernatant 9 Supernatant concentrate 10 Diluent 10a Aqueous layer

Claims (17)

  1. 　3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
    　ことを特徴とする肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
    A liver protective agent, a glucose metabolism improving agent, and an anti-obesity agent.
  2. 　カキ肉から抽出された抽出物に含有する3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
    　ことを特徴とする肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) contained in the extract extracted from oyster meat as an active ingredient,
    A liver protective agent, a glucose metabolism improving agent, and an anti-obesity agent.
  3. 　酢酸エチルを用いてカキ肉から3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)分画を回収し、該回収した3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
    　ことを特徴とする肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          A fraction of 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) was recovered from oyster meat using ethyl acetate, and the recovered 3,5-dihydroxy-4-methoxybenzyl alcohol was recovered. Alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
    A liver protective agent, a glucose metabolism improving agent, and an anti-obesity agent.
  4. 　酢酸エチルとエタノールを用いてカキ肉から3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)分画を回収し、該回収した3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
    　ことを特徴とする肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          3,5-dihydroxy-4-methoxybenzyl alcohol fraction was recovered from oyster meat using ethyl acetate and ethanol, and the recovered 3,5-dihydroxy-4- Methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
    A liver protective agent, a glucose metabolism improving agent, and an anti-obesity agent.
  5. 　抽出用溶液が貯留された抽出容器にカキ肉を収納してカキ肉抽出物入り溶液を採取し、採取されたカキ肉抽出物入り溶液に酢酸エチルを加えて、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)分画を回収し、該回収した3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
    　ことを特徴とする肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          The oyster meat is stored in the extraction container in which the extraction solution is stored, the solution containing the oyster meat extract is collected, ethyl acetate is added to the collected solution containing the oyster meat extract, and 3,5-dihydroxy-4-methoxybenzyl The alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) fraction is recovered, and the recovered 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is used as an active ingredient. ,
    A liver protective agent, a glucose metabolism improving agent, and an anti-obesity agent.
  6. 　抽出用溶液が貯留された抽出容器にカキ肉を収納してカキ肉抽出物入り溶液を採取し、採取されたカキ肉抽出物入り溶液に酢酸エチルとエタノールを加えて、3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)分画を回収し、該回収した3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
    　ことを特徴とする肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          The oyster meat is stored in the extraction container in which the extraction solution is stored, and the solution containing the oyster meat extract is collected. Ethyl acetate and ethanol are added to the collected solution containing the oyster meat extract, and 3,5-dihydroxy-4- The fraction of methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is recovered, and the recovered 3,5-dihydroxy-4-methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) is an active ingredient. And
    A liver protective agent, a glucose metabolism improving agent, and an anti-obesity agent.
  7. 　抽出用溶液が貯留された抽出容器にカキ肉を収納してカキ肉抽出物入り溶液を採取し、採取されたカキ肉抽出物入り溶液にエタノールを加えて、上澄み液と沈殿物とに分離し、前記分離された上澄み液を取り出すとともに、該上澄み液に酢酸エチルを加えて、酢酸エチル層と水層とに分離し、
    　分離した酢酸エチル層の溶液を濃縮して3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)分画を回収し、該回収した3、5-ジヒドロキシ-4-メトキシベンジルアルコール(3、5-dihydroxy-4-methoxybenzyl alcohol)を有効成分とする、
    　ことを特徴とする肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          The oyster meat is stored in the extraction container in which the extraction solution is stored, the solution containing the oyster meat extract is collected, ethanol is added to the collected solution containing the oyster meat extract, and the supernatant liquid and the precipitate are separated. The separated supernatant is taken out, and ethyl acetate is added to the supernatant to separate the ethyl acetate layer and the aqueous layer,
    The separated ethyl acetate layer solution was concentrated to recover 3,5-dihydroxy-4-methoxybenzyl alcohol fraction, and the recovered 3,5-dihydroxy-4 -Methoxybenzyl alcohol (3,5-dihydroxy-4-methoxybenzyl alcohol) as an active ingredient,
    A liver protective agent, a glucose metabolism improving agent, and an anti-obesity agent.
  8. 　前記肝臓保護剤は、肝細胞保護作用を有する、
    　ことを特徴とする請求項1乃至請求項７のいずれかに記載された肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          The hepatoprotective agent has a hepatocyte protective effect,
    A hepatoprotective agent, a glucose metabolism improving agent, and an anti-obesity agent according to any one of claims 1 to 7.
  9. 　前記肝臓保護剤は、抗アポトーシス作用を有する、
    　ことを特徴とする請求項1乃至請求項７のいずれかに記載された肝臓保護剤、糖代謝改善剤及び抗肥満剤。
    　　     The hepatoprotective agent has an anti-apoptotic effect,
    A hepatoprotective agent, a glucose metabolism improving agent, and an anti-obesity agent according to any one of claims 1 to 7.
  10. 　前記肝臓保護剤は、抗脂肪肝作用を有する、
    　ことを特徴とする請求項1乃至請求項７のいずれかに記載された肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          The hepatoprotective agent has an anti-fatty liver effect,
    A hepatoprotective agent, a glucose metabolism improving agent, and an anti-obesity agent according to any one of claims 1 to 7.
  11. 　前記肝臓保護剤は、抗NASH作用を有する、
    　ことを特徴とする請求項1乃至請求項７のいずれかに記載された肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          The hepatoprotective agent has an anti-NASH effect,
    A hepatoprotective agent, a glucose metabolism improving agent, and an anti-obesity agent according to any one of claims 1 to 7.
  12. 　前記肝臓保護剤は、抗炎症作用を有する、
    　ことを特徴とする請求項1乃至請求項７のいずれかに記載された肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          The hepatoprotective agent has an anti-inflammatory effect,
    A hepatoprotective agent, a glucose metabolism improving agent, and an anti-obesity agent according to any one of claims 1 to 7.
  13. 　前記肝臓保護剤は、抗線維化作用を有する、
    　ことを特徴とする請求項1乃至請求項７のいずれかに記載された肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          The hepatoprotective agent has an antifibrotic effect,
    A hepatoprotective agent, a glucose metabolism improving agent, and an anti-obesity agent according to any one of claims 1 to 7.
  14. 　前記糖代謝改善剤は、糖代謝改善作用を有する、
    　ことを特徴とする請求項1乃至請求項７のいずれかに記載された肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          The sugar metabolism improving agent has a sugar metabolism improving action,
    A hepatoprotective agent, a glucose metabolism improving agent, and an anti-obesity agent according to any one of claims 1 to 7.
  15. 　前記抗肥満剤は、抗肥満作用を有する、　ことを特徴とする請求項1乃至請求項７のいずれかに記載された肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          The liver protective agent, the glucose metabolism improving agent, and the anti-obesity agent according to any one of claims 1 to 7, wherein the anti-obesity agent has an anti-obesity action.
  16. 　前記肝臓保護剤は、肝臓におけるDNA、脂質、及びタンパク質における抗酸化作用を有する、
    　ことを特徴とする請求項1乃至請求項７のいずれかに記載された肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          The hepatoprotectant has an antioxidant action on DNA, lipids, and proteins in the liver,
    A hepatoprotective agent, a glucose metabolism improving agent, and an anti-obesity agent according to any one of claims 1 to 7.
  17. 　前記肝臓保護剤は、インスリン抵抗性改善作用を有する、
    　ことを特徴とする請求項1乃至請求項７のいずれかに記載された肝臓保護剤、糖代謝改善剤及び抗肥満剤。
          The hepatoprotectant has an insulin resistance improving action,
    A hepatoprotective agent, a glucose metabolism improving agent, and an anti-obesity agent according to any one of claims 1 to 7.
PCT/JP2015/003141 2014-07-15 2015-06-23 Hepatoprotective agent, glucose metabolism-improving agent, and anti-obesity agent WO2016009594A1 (en)

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JP2018118910A (en) * 2017-01-23 2018-08-02 株式会社渡辺オイスター研究所 Oyster extract having sleep improvement action based sleep efficiency deterioration suppressing action, sleep difficulty improvement action, and sleep related matters for quality of life (hereinafter referred to as qol), and having action for suppressing increase in awakening mid-sleep by maintaining total arousal frequency
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TWI814228B (en) * 2021-11-30 2023-09-01 日商渡邊牡蠣研究所股份有限公司 Proliferation Inhibitor of Metastatic Human Prostate Cancer Cells
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